Methods and drug products for treating alzheimer&#39;s disease

ABSTRACT

Provided herein are drug products with low dose pioglitazone for use in the treatment (e.g., delay of onset) of cognitive impairment of the Alzheimer&#39;s type. Methods of manufacture thereof are also provided. Further provided are methods of treatment for Alzheimer&#39;s disease including administering a drug product with low dose pioglitazone. The methods may include determining whether the subject is at risk of developing Alzheimer&#39;s disease based upon the subject&#39;s age and TOMM40 523 genotype.

RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.13/346,081, which claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Patent Application No. 61/431,370, filed Jan. 10, 2011.

FIELD OF THE INVENTION

The present invention relates to a method and drug product for treatinga subject who is at risk to develop Alzheimer's disease.

BACKGROUND

Alzheimer's disease is a neurodegenerative disease and the most commoncause of dementia. This disease manifests as a gradual but progressivedecline in memory, thinking skills and behavior that is acceleratedrelative to normal aging (Reitz et al. 2011 Nat Rev Neurol 7: 137-152).Eventually, patients are unable to recognize familiar people or carryout the simplest task. Alzheimer's disease is, at this time, the sixthleading cause of death in the United States (US).

There are two predominant forms of the disease: Familial Alzheimer'sdisease is typically caused by dominant mutations in one of three genes(APP, PSEN1 or PSEN2). This form of the disease is a rare anddevastating illness with onset occurring in mid-life. The second and farmore common form of the disease is Sporadic or Late onset Alzheimer'sdisease (hereinafter “Alzheimer's disease” or “AD”). Onset ofAlzheimer's disease typically occurs after the age of 62 years.

As the world population and human longevity increase, so do the numbersof people affected by Alzheimer's disease globally. The estimatedworldwide costs of dementia, of which Alzheimer's disease accounts forup to 80% of cases, was US$604 billion in 2010, which was greater than1% of US GDP (Wimo and Prince 2010 World Alzheimer Report 2010: TheGlobal Economic Impact of Dementia 1-93). The cost of caring forAlzheimer patients in the US is expected to increase from US$172 millionin 2010, to US$1.07 trillion in 2050 (Alzheimer's Association. “Changingthe Trajectory of Alzheimer's Disease: A National Imperative (2010)”).

At this time, the few drugs that are approved for treatment of thisdisease provide some symptomatic relief, but this is typically ofrelatively short duration, and the therapies do not alter the course ofdisease progression (Alzheimer's Association. “Changing the Trajectoryof Alzheimer's Disease: A National Imperative (2010)”). Therapies thatdelay the onset of the disease, reduce the rate of disease progression,or that can do both are urgently needed. Therapies that can achieveeither of these goals will reduce the number of individuals withdisease, or reduce the number of individuals with the more advanced anddebilitating stages of disease (Brookmeyer et al. 2007 Alzheimers Dement3: 186-191). It is projected that if the onset of Alzheimer's disease isdelayed by 5 years due to availability of a breakthrough therapy in2015, 43% of the 13.5 million Americans expected to have the conditionin 2050 would not have the disease, and there will be fewer people withadvanced disease.

The principal risk factor for Alzheimer's disease is age, and prevalenceof the disease increases with age (approximately 10% of individuals over65 and approximately 50% of individuals over 85). The incidence of thedisease doubles every 5 years after 65 years of age, with the diagnosisof about 1275 new cases per year per 100,000 persons older than 65 yearsof age (Querfurth et al., 2010 NEJM 362:4). Both men and women areaffected by Alzheimer's disease, but women generally represent a higherpercentage of cases overall (roughly 60% to 40%), possibly due togreater longevity. People suffering from Alzheimer's disease tend tolive approximately 3 to 9 years after diagnosis, on average.

The epsilon 4 allele of APOE has previously been associated withincreased risk of developing Alzheimer's disease. (Pericak-Vance et al.1991 Am J Hum Genet 48: 1034-1050; Martin et al. 2000 Am J Hum Genet 67:383-394; U.S. Pat. Nos. 6,027,896 and 5,716,828 to Roses et al.) Therelationship is copy number dependent (Yoshizawa et al. 1994 Ann Neurol36: 656-659). That is to say, a carrier of two APOE4 alleles is morelikely to develop late-onset Alzheimer's disease (LOAD) than a carrierof only one APOE4 allele, and at an earlier age (Corder et al. 1993Science 261, 921-3).

Nevertheless, APOE4 alleles only account for roughly 50% of theinherited risk of late onset Alzheimer's disease. One explanation isthat APOE4 is merely serving as a surrogate marker for something inlinkage disequilibrium nearby. Alternatively, considering the recentdiscovery of a mechanistic role for APOE4 in mitochondrial toxicity, thenegative effects of APOE4 may be abrogated or exacerbated by anothergene product that may be encoded nearby (Chang et al. 2005 Proc NatlAcad Sci USA 102: 18694-18699).

The symptoms of Alzheimer's disease are primarily marked by cognitivedeficits including memory impairment, language dysfunction, andvisuospatial skills; functional impairment that may span occupationaland social issues (e.g., activities of daily living); and behavioralsymptoms including depression, anxiety, aggression and psychosis mayalso appear as the disease progresses in severity.

At this time, unambiguous diagnosis of Alzheimer's disease requiresclinical findings of cognitive deficits consistent with AD andpost-mortem identification of brain pathologies consistent with AD. Theterm AD dementia is used to describe dementia that is due to thepathophysiologies of Alzheimer's disease. The term “probable Alzheimer'sdisease” is used in life when a subject demonstrates clinicalcharacteristics of Alzheimer's disease and when other possiblebiological causes of dementia (e.g. Parkinson's disease or stroke) areexcluded.

There are currently a variety of art-accepted methods for diagnosingprobable Alzheimer's disease. Typically, these methods are used incombination. These methods include determining an individual's abilityto carry out daily activities and identifying changes in behavior andpersonality. Dementia of the AD type is also typically characterized byan amnestic presentation (memory deficit) or language, visuospatial orexecutive function deficits. Cognitive ability/impairment may bedetermined by art-accepted methods, including, but not limited to,validated instruments that assess global cognition (e.g., the ModifiedMini Mental State Examination (3MS-E)), and specific domains such asvisual and verbal memory (e.g., the Brief Visuospatial Memory Test(Revised) (BVMT-R) and the Hopkins Verbal Learning Test (Revised)(HVLT-R), respectively), language (e.g., the Generative Verbal FluencyTest (GVFT)) and executive function and attention (e.g., the Digit SpanTest (DST)). Dementia due to AD is also defined by insidious onset and ahistory of worsening cognitive performance.

The criteria for ‘probable Alzheimer's disease’ were recently updated bya National Institute of Aging-Alzheimer's Association workgroup (McKhannet al. 2011 Alzheimers Dement 7: 263-269). This workgroup recommendedthat, for people who first exhibit the core clinical characteristics ofAlzheimer's disease dementia, evidence of biomarkers associated with thedisease may enhance the certainty of the diagnosis.

In view of the fact that more than 4.5 million people in the UnitedStates alone suffer from Alzheimer's disease (and this number willcontinue to grow as the population ages), the cruel and unforgivingdegenerative and debilitative nature of Alzheimer's disease as itdevelops, and the high costs associated with the care for peoplesuffering from Alzheimer's disease, there is a real and immediate needfor an effective medical therapy that can delay the onset of Alzheimer'sdisease.

BRIEF SUMMARY OF THE INVENTION

Provided herein are compositions including low dose pioglitazone, whichcompositions are useful in treating mild cognitive impairment (e.g.,cognitive impairment of the Alzheimer's type). In some embodiments,treating includes delaying the onset of mild cognitive impairment. Insome embodiments, treating includes delaying the onset of mild cognitiveimpairment in a cognitively normal subject. In some embodiments, thedelaying includes delaying the onset of impairment in episodic memory.

In some embodiments, treating includes delaying the onset of mildcognitive impairment in a human subject at increased risk of developingcognitive impairment within the next 5-7 years, said risk based upon thesubject's age, or based upon the subject's age and TOMM40 rs10524523genotype.

In some embodiments, low dose pioglitazone is administered in unitdosage form, e.g., having from 0.5, 1, 1.5 or 2, to 6, 8, 10 or 12milligrams of pioglitazone or a pharmaceutically acceptable saltthereof.

Also provided is the use of low dose pioglitazone in the manufacture ofa pharmaceutical formulation for the treatment of mild cognitiveimpairment (e.g., cognitive impairment of the Alzheimer's type). In someembodiments, the pharmaceutical formulation is a tablet. In someembodiments, the pharmaceutical formulation is a capsule. In someembodiments, the pharmaceutical formulation is a caplet. In someembodiments, the pharmaceutical formulation is a liquid. In someembodiments, the pharmaceutical formulation is a solid or semi-solid.

Also provided is a composition including low dose pioglitazone for usein the treatment of cognitive decline.

Further provided are methods for treating mild cognitive impairment(e.g., cognitive impairment of the Alzheimer's type) in a human subjectin need thereof, comprising administering to the subject low dosepioglitazone. In some embodiments, the treating includes delaying theonset of mild cognitive impairment. In some embodiments, treatingincludes delaying the onset of mild cognitive impairment in acognitively normal subject. In some embodiments, the delaying includesdelaying the onset of impairment in episodic memory.

In some embodiments, the subject is at increased risk in developingcognitive impairment of the Alzheimer's type within the next 5-7 years,said risk based upon the subject's age, or based upon the subject's ageand rs10524523 (′523) genotype.

In some embodiments, the subject is at least 50, 55, 60, 62, 68, or 70years old.

In some embodiments, the subject is a Caucasian subject. In someembodiments, the subject is a non-Caucasian subject.

In some embodiments, the subject does not have one or two APOE2 alleles.

In some embodiments, low dose pioglitazone is administered in unitdosage form, e.g., having from 0.5, 1, 1.5 or 2, to 6, 8, 10 or 12milligrams of pioglitazone. In some embodiments, the administering isonce daily.

In some embodiments, pioglitazone is provided as or administered at adosage that provides an AUC of from about 0.15 μg·h/mL to about 3.6μg·h/mL. In some embodiments, pioglitazone is provided as oradministered at a dosage that provides an AUC of from 0.12 μg·h/mL to4.5 μg·h/mL. In some embodiments, pioglitazone is provided as oradministered at a dosage that provides an AUC of from 0.12 μg·h/mL to3.4 μg·h/mL.

Also provided are methods of treating cognitive decline in a humansubject in need thereof, including administering to said subject lowdose pioglitazone.

Still further provided are methods of determining increased risk indeveloping cognitive impairment of the Alzheimer's type in a humansubject at a predetermined age or age range, including:

detecting from a biological sample of said subject the ′523 genotype ofsaid subject, wherein each allele of ′523 is assigned as:

-   -   (a) short (S, less than 19 T residues);    -   (b) long (L, 19-29 residues); or    -   (c) very long (VL, 30 or more residues); and

determining from said ′523 genotype whether said subject is at increasedrisk in developing cognitive impairment of the Alzheimer's type at saidpredetermined age or age range, wherein:

(1) age greater than about 62 and L,L or L,VL indicates increased risk;

(2) age greater than about 62 and VL,VL does not indicate increasedrisk;

(3) age greater than about 74 and S,L indicates increased risk;

(4) age greater than about 77 and S,S indicates increased risk; and

(5) age greater than about 76 and S,VL indicates increased risk.

In some embodiments, the determining further includes detecting from abiological sample of said subject the APOE genotype of said subject,wherein the presence of an APOE2 allele in said genotype indicates thesubject is not at increased risk.

Also provided are methods of determining whether to administer low dosepioglitazone to a human subject for treatment of cognitive impairment ofthe Alzheimer's type, including:

detecting from a biological sample of said subject the ′523 genotype ofthe subject, wherein each allele is assigned as:

-   -   (a) short (S, less than 19 T residues);    -   (b) long (L, 19-29 residues); or    -   (c) very long (VL, 30 or more residues); and

determining from said ′523 genotype and from the age of said humansubject whether to administer low dose pioglitazone to said subject fortreatment of cognitive impairment of the Alzheimer's type, wherein:

(1) age greater than about 62 and L,L or L,VL indicates treatment;

(2) age greater than about 62 and VL,VL does not indicate treatment;

(3) age greater than about 74 and S,L indicates treatment;

(4) age greater than about 77 and S,S indicates treatment; and

(5) age greater than about 76 and S,VL indicates treatment.

In some embodiments, the determining further includes detecting from abiological sample of said subject the APOE genotype of said subject,wherein the presence of an APOE2 allele in said genotype does notindicate treatment.

In some embodiments of any of the above methods or compositions, thesubject has normal cognition.

Still further provided are methods of delaying the onset of Alzheimer'sdisease, wherein the method comprises (a) detecting a variant to theTOMM40 gene in a subject who is at-risk to develop Alzheimer's disease,and (b) administering a drug product that contains an effective low dosepioglitazone or pioglitazone salt to the at-risk subject detected withthe TOMM40 variant to delay the onset of Alzheimer's disease. Forexample, the present invention contemplates (a) detecting a variant ofthe TOMM40 gene, such as a long poly-T allele (greater than 19 Thymidineresidues), in a subject who is at-risk to develop Alzheimer's disease,and (b) administering an effective amount of low dose pioglitazone orpioglitazone salt drug product o the at-risk subject detected with thelong poly-T allele variant of the TOMM40 gene, who may for example be ina normal cognitive stage, to delay the onset of Alzheimer's disease.

Also provided are methods of delaying the onset of one or more stagesthat progress to Alzheimer's disease, such as the mild cognitiveimpairment stage, the amnestic mild cognitive impairment stage, thepreclinical Alzheimer's disease stage and/or the prodromal Alzheimer'sdisease stage, in a subject at-risk to develop Alzheimer's disease,wherein the method comprises: (a) detecting in a subject who is at-riskto develop Alzheimer's disease a variant to the TOMM40 gene, such as along poly-T allele (greater than 19 Thymidine residues); and (b)administering a drug product that contains an effective amount of lowdose pioglitazone or pioglitazone salt to the at-risk subject in whomthe TOMM40 variant has be detected to delay the onset of one or more ofthe stages that progress to Alzheimer's disease, including any cognitiveimpairment or other stage, to delay the onset of Alzheimer's disease inthe at-risk subject. It should be understood that, in accordance withthis method of the present invention, the at-risk subject, at time ofdetection of the TOMM40 variant and/or treatment, may be in a normalcognitive stage or in any one of the stages that progress to Alzheimer'sdisease.

The above summary is not intended to describe each disclosed embodimentor every implementation of the present invention. The description thatfollows more particularly exemplifies illustrative embodiments. Inseveral places throughout the application, guidance is provided throughlists of examples, which examples can be used in various combinations.In each instance, the recited list serves only as a representative groupand should not be interpreted as an exclusive list.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents fMRI images of rat brain at multiple doses of PIOrelative to vehicle control. The top panel shows the group-averaged fMRIsignal at baseline; the bottom panel illustrates the group-averaged fMRIsignal at treatment day 7. This analysis shows that pioglitazone HCl atdoses as low as 0.04 mg/kg/day induces change in metabolism in deepsubcortical structure of the rat brain.

FIG. 2 presents a graph of the age at onset of cognitive impairment ofthe Alzheimer type for each of the TOMM40 523 genotypes. The Y axisshows the percent survival without cognitive impairment, while the Xaxis represents age. Data obtained from the Duke Bryan ADRC cohort N=438subjects, 106 diagnosed with cognitive impairment, 332 cognitivelynormal. N for each genotype: L,L:23; L,VL:54; S,L:72; S,S:100; S,VL:138;VL,VL:51.

FIG. 3 presents the curve showing the age at onset of cognitiveimpairment of the Alzheimer type for individuals possessing the S,L 523genotype. The Y axis shows the percent survival without cognitiveimpairment, while the X axis represents age. The curve shows a steepslope beginning at age 74 (vertical line). Individuals entering thetrial at or above age 74 who possess the S,L 523 genotype are at highrisk of developing cognitive impairment during the next 5 years. Data isobtained from the Duke Bryan ADRC cohort, N=72 subjects, 23 diagnosedwith cognitive impairment, 49 cognitively normal.

FIG. 4 presents the curve showing the age at onset of cognitiveimpairment of the Alzheimer's type for 523 L,L genotype. The Y axisshows the percent survival without Cl, while the X axis represents age.Data obtained from the Duke Bryan ADRC cohort N=23 subjects, 11diagnosed with Cl, 12 cognitively normal.

FIG. 5 presents the curve showing age at onset of cognitive impairmentof the Alzheimer's type for 523 L,VL genotype. The Y axis shows thepercent survival without Cl, while the X axis represents age. Dataobtained from the Duke Bryan ADRC cohort N=54 subjects, 24 diagnosedwith Cl, 30 cognitively normal.

FIG. 6 presents the curve showing age at onset of cognitive impairmentof the Alzheimer's type for 523 S,L genotype. The Y axis shows thepercent survival without Cl, while the X axis represents age. Dataobtained from the Duke Bryan ADRC cohort N=72 subjects, 23 diagnosedwith Cl, 49 cognitively normal.

FIG. 7 presents the curve showing age at onset of cognitive impairmentof the Alzheimer's type for 523 S,S genotype. The Y axis shows thepercent survival without Cl, while the X axis represents age. Dataobtained from the Duke Bryan ADRC cohort N=100 subjects, 20 diagnosedwith Cl, 80 cognitively normal.

FIG. 8 presents the curve showing age at onset of cognitive impairmentof the Alzheimer's type for 523 S,VL genotype. The Y axis shows thepercent survival without Cl, while the X axis represents age. Dataobtained from the Duke Bryan ADRC cohort N=138 subjects, 22 diagnosedwith Cl, 116 cognitively normal.

FIG. 9 presents the curve showing age at onset of cognitive impairmentof the Alzheimer's type for 523 VL,VL genotype. The Y axis shows thepercent survival without Cl, while the X axis represents age. Dataobtained from the Duke Bryan ADRC cohort N=51 subjects, 6 diagnosed withCl, 45 cognitively normal.

DETAILED DESCRIPTION OF THE INVENTION

By way of illustrating and providing a more complete appreciation of thepresent invention and many of the attendant advantages thereof, thefollowing detailed description and examples are given concerning thenovel methods and compositions.

In one aspect, the present invention relates to a pharmaceuticalcomposition, i.e., a drug product, comprising low dose pioglitazone or apharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable vehicle for administration to a subject, such as a humanpatient in need of treatment to delay the onset of or otherwise treatAlzheimer's disease in such a patient. While the present invention maybe embodied in many different forms, several specific embodiments arediscussed herein with the understanding that the present disclosure isto be considered only as an exemplification of the principles of theinvention, and it is not intended to limit the invention to theembodiments described or illustrated.

I. DEFINITIONS

As used in the description of the invention and the appended claims, thesingular forms “a”, “an” and “the” are used interchangeably and intendedto include the plural forms as well and fall within each meaning, unlessthe context clearly indicates otherwise. Also, as used herein, “and/or”refers to and encompasses any and all possible combinations of one ormore of the listed items, as well as the lack of combinations wheninterpreted in the alternative (“or”).

As used herein, “at least one” is intended to mean “one or more” of thelisted elements.

Singular word forms are intended to include plural word forms and arelikewise used herein interchangeably where appropriate and fall withineach meaning, unless expressly stated otherwise.

Except where noted otherwise, capitalized and non-capitalized forms ofall terms fall within each meaning.

Unless otherwise indicated, it is to be understood that all numbersexpressing quantities, ratios, and numerical properties of ingredients,reaction conditions, and so forth used in the specification and claimsare contemplated to be able to be modified in all instances by the term“about”.

All parts, percentages, ratios, etc. herein are by weight unlessindicated otherwise.

As used herein, “bioequivalence” or “bioequivalent”, refers to low dosepioglitazone formulations or drug products which are pharmaceuticallyequivalent, and their bioavailabilities (rate and extent of absorption)after administration in the same molar dosage or amount are similar tosuch a degree that their therapeutic effects, as to safety and efficacy,are essentially the same. In other words, bioequivalence orbioequivalent means the absence of a significant difference in the rateand extent to which pioglitazone becomes available from suchformulations at the site of pioglitazone action when administered at thesame molar dose under similar conditions, e.g., the rate at whichpioglitazone can leave such a formulation and the rate at whichpioglitazone can be absorbed and/or become available at the site ofaction to affect Alzheimer's disease. In other words, there is a highdegree of similarity in the bioavailabilities of two pioglitazonepharmaceutical products (of the same galenic form) from the same molardose, that are unlikely to produce clinically relevant differences intherapeutic effects, or adverse reactions, or both. The terms“bioequivalence”, as well as “pharmaceutical equivalence” and“therapeutic equivalence” are also used herein as defined and/or used by(a) the United States Food and Drug Administration (FDA), (b) the Codeof Federal Regulations (“C.F.R.”), Title 21, (c) Health Canada, (d)European Medicines Agency (EMEA), and/or (e) the Japanese Ministry ofHealth and Welfare. Thus, it should be understood that the presentinvention contemplates low dose pioglitazone formulations or drugproducts that may be bioequivalent to other low dose pioglitazoneformulations or drug products of the present invention. By way ofexample, a first low dose pioglitazone formulation or drug product isbioequivalent to a second low dose pioglitazone formulation or drugproduct, in accordance with the present invention, when the measurementof at least one pharmacokinetic parameter(s), such as a Cmax, Tmax, AUC,etc., of the first low dose pioglitazone formulation or drug productvaries by no more than about ±25%, when compared to the measurement ofthe same pharmacokinetic parameter for the second low dose pioglitazoneformulation or drug product.

As used herein, “bioavailability” or “bioavailable” means generally therate and extent of absorption of pioglitazone into the systemiccirculation and, more specifically, the rate or measurements intended toreflect the rate and extent to which pioglitazone becomes available atthe site of action or is absorbed from a drug product and becomesavailable at the site of action. In other words, and by way of example,the extent and rate of pioglitazone absorption from a lower dosagestrength formulation of the present invention as reflected by atime-concentration curve of pioglitazone in systemic circulation.

By way of further example, bioavailability is a measurement of theextent of a therapeutically active drug that reaches the systemiccirculation and is available at the site of action. It is expressed asthe letter F.

With respect to absolute bioavailability, absolute bioavailabilitycompares the bioavailability (estimated as area under the curve, or AUC)of the active drug in systemic circulation following non-intravenousadministration (i.e., after oral, rectal, transdermal, subcutaneousadministration), with the bioavailability of the same drug followingintravenous administration. It is the fraction of the drug absorbedthrough non-intravenous administration compared with the correspondingintravenous administration of the same drug. The comparison must be dosenormalized if different doses are used; consequently, each AUC iscorrected by dividing the corresponding dose administered.

In order to determine absolute bioavailability of a drug, apharmacokinetic study must be done to obtain a plasma drug concentrationvs time plot for the drug after both intravenous (IV) andnon-intravenous administration. The absolute bioavailability is thedose-corrected area under curve (AUC) non-intravenous divided by AUCintravenous. For example, the formula for calculating F for a drugadministered by the oral route (po) is given below.

$F = \frac{\lbrack{AUC}\rbrack_{po}*{dose}_{IV}}{\lbrack{AUC}\rbrack_{IV}*{dose}_{po}}$

Therefore, a drug given by the intravenous route will have an absolutebioavailability of 1 (F=1) while drugs given by other routes usuallyhave an absolute bioavailability of less than one.

With respect to relative bioavailability, this measures thebioavailability (estimated as area under the curve, or AUC) of a certaindrug when compared with another formulation of the same drug, usually anestablished standard, or through administration via a different route.When the standard consists of intravenously administered drug, this isknown as absolute bioavailability.

${{relative}\mspace{14mu} {bioavailability}} = \frac{\lbrack{AUC}\rbrack_{A}*{dose}_{B}}{\lbrack{AUC}\rbrack_{B}*{dose}_{A}}$

As used herein, the terms “pharmaceutical equivalence” or“pharmaceutically equivalent” refer to low dose pioglitazoneformulations or drug products of the present invention that contain thesame amount of pioglitazone, in the same dosage forms, but notnecessarily containing the same inactive ingredients, for the same routeof administration and meeting the same or comparable compendial or otherapplicable standards of identity, strength, quality, and purity,including potency and, where applicable, content uniformity and/orstability. Thus, it should be understood that the present inventioncontemplates low dose pioglitazone formulations or drug products thatmay be pharmaceutically equivalent to other low dose pioglitazoneformulations or drug products used in accordance with the presentinvention.

As used herein, the terms “therapeutic equivalence or therapeuticallyequivalent” mean those low dose pioglitazone formulations or drugproducts which (a) will produce the same clinical effect and safetyprofile when utilizing pioglitazone drug product to delay onset ofAlzheimer's disease in accordance with the present invention and (b) arepharmaceutical equivalents, e.g., they contain pioglitazone in the samedosage form, they have the same route of administration; and they havethe same pioglitazone strength. In other words, therapeutic equivalencemeans that a chemical equivalent of a lower dosage strength pioglitazoneformulation of the present invention (i.e., containing the same amountof pioglitazone in the same dosage form when administered to the sameindividuals in the same dosage regimen) will provide essentially thesame efficacy and toxicity.

“Alzheimer's disease”, “Alzheimer disease”, or “AD” as used herein is adisease in which cognitive function is impaired gradually over time, andincludes a symptomatic pre-dementia phase with presentation of mildcognitive impairment (MCI), and a dementia phase, where there is asignificant impairment in social or occupational functioning. See Albertet al. 2011 Alzheimer's & Dementia 7: 270-279; McKhann et al. 2011Alzheimer's & Dementia 7: 263-269.

Though a number of biomarkers are reported to coincide with Alzheimer'sdisease, none are recognized as validated or qualified biomarkers forthe diagnosis or prognosis of Alzheimer's disease by the US Food andDrug Administration. From a clinical standpoint, the hallmark featurethat is consistently present and needed for the diagnosis of Alzheimer'sdisease is cognitive impairment.

Indications of cognitive impairment may include, but are not limited to,difficulty with mental functions such as language, memory (e.g.,episodic), perception, emotional behavior or personality, cognitiveskills (e.g., calculation, abstract thinking, judgment). Thedetermination may be obtained from the patient, from an informant whoknows the patient well, from a skilled clinician observing the patient,or a combination thereof.

“Mild cognitive impairment” or “MCI” refers to a reduction in cognitiveability that is greater than anticipated considering a person's age oreducation in one or more cognitive domains. The cognitive domainsinclude memory, executive functions (e.g., problem-solving, planning orreasoning), attention (e.g., simple and divided attention), visuospatialskill, and language (e.g., naming, fluency, expressive speech,comprehension). Symptoms of MCI may include difficulties identifying theright word or name; difficulty remembering names when introduced to newpeople; noticeably greater difficulty performing tasks in social or worksettings; forgetting material that one has just read; losing ormisplacing a valuable object; increasing trouble with planning ororganizing; difficulty mastering new skills; concentration deficits; andincreased anxiety. Mild cognitive impairment is a phase at whichsymptoms are sufficient to meet the currently accepted criteria of MCI,but where symptoms do not meet dementia diagnostic criteria. People withMCI, however, may remain functionally intact and independent. If formal,standardized cognitive tests are administered, people with MCI generallyscore 1 to 1.5 standard deviations below the age and education-adjustedmean for their peers. It should be noted that not all MCI leads todementia, nor to Alzheimer's disease.

“Cognitive Impairment of the Alzheimer's Type” or “CIAT” as used hereinrefers to cognitive impairment consistent with features whereinAlzheimer's is the likely cause, and thus may be considered a subset ofMCI. The designations, “Cognitive Impairment of the Alzheimer's Type”,“Mild Cognitive Impairment due to Alzheimer's disease (MCI due to AD)”or “amnestic Mild Cognitive Impairment (aMCI)” refer to the symptomatic,pre-dementia phase of Alzheimer's disease. CIAT or MCI due to AD isdetermined following use of neuropsychological tests and clinicianassessment of the cognitive function of the individual. Typically,episodic memory is impaired in person with MCI that progresses to AD(aMCI). However, there are atypical forms of MCI—MCI with nonamnesticpresentation—that also progress to Alzheimer's disease. Progressivedecline in cognitive function provides additional evidence that a personsuffers MCI due to AD.

There are a number of neuropsychological assessments, particularly thosethat test episodic memory (i.e., the ability to learn and retain newinformation), that are useful in diagnosing MCI due to AD, or thosepatients with MCI who are likely to progress to AD within a few years.Tests of episodic memory may assess immediate and/or delayed recall,such as word-list learning tests. In addition, an alternative etiologyfor the cognitive impairment, such as degenerative (e.g., Parkinsonism),vascular events including microinfarcts, depressive, traumatic, medicalcomorbidities, should be ruled out. A number of biomarkers have beenproposed for use in research and may also be useful in supporting theclinical diagnosis of MCI due to AD by confirming the presence ofpathologies consistent with AD or to monitor progression of the disease,if desired. See, e.g., Albert et al. 2011 Alzheimer's & Dementia 7:270-279.

In accordance with the present invention, cognitive impairment may bedetermined by any art-accepted method of cognitive assessment,including, but not limited to, an assessment of global cognition (e.g.,the Modified Mini Mental State Examination (3MS-E)), and specificdomains such as visual and verbal memory (e.g., the Brief VisuospatialMemory Test (Revised) (BVMT-R) and the Hopkins Verbal Learning Test(Revised) (HVLT-R), respectively), language (e.g., the Generative VerbalFluency Test (GVFT)) and executive function and attention (e.g., theDigit Span Test (DST)).

Physiological changes may or may not also be detected. “Physiologicalchanges” means, for example, the occurrence of at least one of alteredfunctional connectivity, brain atrophy, decreased synaptic activity inthe brain, increased amyloid accumulation in the brain, decreasedmitochondrial function or increased mitochondrial dysfunction in thebrain, neuronal formation of neurofibrillary tangles in the brain, and achange corresponding to any other symptom of Alzheimer's disease.Physiological changes that can be indicative of Alzheimer's diseaseinclude, but are not limited to, hypometabolism in the brain, alteredfunctional connectivity, increased beta amyloid in the brain and or CSFand tau and phospho-tau in the CSF.

As used herein, “onset” means the occurrence in a subject of clinicalsymptoms associated or consistent with a diagnosis Alzheimer's diseaseor a phase that progresses to Alzheimer's dementia, such as CIAT, asdefined herein.

As used herein, “delay” in the onset or progression of a phaseconsistent with Alzheimer's disease means an increase in time from afirst time point to onset or worsening of a phase consistent withAlzheimer's disease, such as cognitive impairment of the Alzheimer type.For example, a delay in the onset of Alzheimer's disease means that theonset of Alzheimer's disease, as defined herein, in a subject at risk todevelop Alzheimer's disease is delayed from happening at its naturaltime frame by at least six months, 1 year, 1½ years, 2, years, 2½ years,3 years, 3½ years, 4 years, 4½ years, 5 years, 5½ years, 6 years, 6½years, 7 years, 7½ years or 8 years or more, and preferably from 3 yearsto 8 years and more preferably for 5 years after a normal cognitivesubject has been determined to be at high risk to develop Alzheimer'sdisease. By way of further example, a delay in the progression ofcognitive impairment that may progress to Alzheimer's disease or a delayin the progression of dementia means that the rate of cognitive declineis slowed relative to its natural time frame. These determinations areperformed by using appropriate statistical analysis.

A “first time point’ includes, for example, the initiation of low dosepioglitazone treatment as taught herein.

In some embodiments, a delay in the onset of cognitive impairmentconsistent with Alzheimer's disease can be determined by, for example,performing any of the cognitive assessments described herein or bymeeting accepted diagnostic criteria for cognitive impairment of theAlzheimer's type. In addition to the assessment of cognitiveperformance, changes in other biomarkers that are consistent withAlzheimer's disease pathologies may also be measured, if desired,including the rate of brain atrophy, for example measured by magneticresonance imaging (MRI) or measurement of the changes in functionalconnections between brain regions, assessment of brain metabolism orneuronal activity, amyloid accumulation in the brain, brain physiologyas measured by BOLD-fMRI signal, mitochondrial function in the brain,mitochondrial proliferation in the brain, diseased neurons,neurofibrillary tangles in the brain, amyloid in the CSF and Tau orphospho-Tau in the CSF, etc.

“Diagnosis” or “prognosis” as used herein refer to the use ofinformation (e.g., genetic information or data from other moleculartests, biological or chemical information from biological samples, signsand symptoms, physical exam findings, cognitive performance results,etc.) to anticipate the most likely outcomes, timeframes, and/orresponses to a particular treatment for a given disease, disorder, orcondition, based on comparisons with a plurality of individuals sharingcommon nucleotide sequences, symptoms, signs, family histories, or otherdata relevant to consideration of a patient's health status, or theconfirmation of a subject's affliction, e.g., with mild cognitiveimpairment (MCI) (e.g., cognitive impairment of the Alzheimer's type).

“Biological sample,” as used herein, refers to a material containing,for example, a nucleic acid, protein or other biological or chemicalmaterial of interest. Biological samples containing nucleic acid such asDNA include hair, skin, cheek swab, and biological fluids such as blood,serum, plasma, sputum, lymphatic fluid, semen, vaginal mucus, feces,urine, spinal fluid, and the like. Isolation of DNA from such samples iswell known to those skilled in the art.

A “subject” according to some embodiments is an individual whosegenotype(s) or haplotype(s) are to be determined and recorded inconjunction with the individual's condition (i.e., disease or disorderstatus) and/or response to a candidate drug or treatment.

“Subject,” as used herein, is preferably, but not necessarily limitedto, a human subject. The subject may be male or female and may be of anyrace or ethnicity, including, but not limited to, Caucasian,African-American, African, Asian, Hispanic, Indian, etc. The subject maybe of any age, including newborn, neonate, infant, child, adolescent,adult, and geriatric. Subject as used herein may also include an animal,particularly a mammal such as a canine, feline, bovine, caprine, equine,ovine, porcine, rodent (e.g., a rat and mouse), a lagomorph, a primate(including non-human primate), etc., that may be treated in accordancewith the methods of the present invention or screened for veterinarymedicine or pharmaceutical drug development purposes. A subjectaccording to some embodiments of the present invention include apatient, human or otherwise, in need of therapeutic treatment to delayonset of Alzheimer's disease.

“Gene,” as used herein, means a segment of DNA that contains informationfor the regulated biosynthesis of an RNA product, including promoters,exons, introns, and other untranslated regions that control expression.

A “genetic risk factor,” as used herein, means a genetic marker that isassociated with increased susceptibility to a condition, disease, ordisorder. It may also refer to a genetic marker that is associated witha particular response to a selected drug or treatment of interest.“Associated with” as used herein means the occurrence together of two ormore characteristics more often than would be expected by chance alone.An example of associated with involves a feature on the surface of whiteblood cells called HLA (HLA stands for human leukocyte antigen). Aparticular HLA type, HLA type B-27, is associated with an increased riskfor a number of diseases including ankylosing spondylitis. Ankylosingspondylitis is 87 times more likely to occur in people with HLA B-27than in the general population.

A “prognostic” marker may be used to predict the probable course of acondition or disease, including, but not limited to, prediction of theprobable age of onset of the condition or disease, course and/or rate ofprogression of the condition or disease, etc. It could include genotypeand/or other variables, including age of the subject.

A subject “at increased risk of developing a condition” due to a geneticrisk factor is one who is predisposed to the condition, has geneticsusceptibility for the condition, and/or is more likely to develop thecondition than subjects in which the genetic risk factor is absent. Asubject “at increased risk” may also be a subject who is susceptible todeveloping the disease at an earlier age.

As used herein, a subject “at-risk of developing Alzheimer's disease”includes an individual that is more likely to develop Alzheimer'sdisease based on one or more of: age; rs10524523 genotype; APOEgenotype, etc.

“Polymorphism,” as used herein, refers to the existence of two or moredifferent nucleotide sequences at a particular locus in the DNA of thegenome. Polymorphisms can serve as genetic markers and may also bereferred to as genetic variants. Polymorphisms include nucleotidesubstitutions, insertions, deletions and microsatellites, and may, butneed not, result in detectable differences in gene expression or proteinfunction. A polymorphic site is a nucleotide position within a locus atwhich the nucleotide sequence varies from a reference sequence in atleast one individual in a population.

A “deletion/insertion polymorphism” or “DIP,” as used herein, is aninsertion of one or more nucleotides in one version of a sequencerelative to another. If it is known which of the alleles represent minoralleles, the term “deletion” is used when the minor allele has adeletion of one or more nucleotides, and the term “insertion” is usedwhen the minor allele has an additional one or more nucleotides. Theterm “deletion/insertion polymorphism” is also used when there aremultiple forms or lengths and it is not apparent which is the minorallele. For example, for the poly-T polymorphisms described herein,multiple lengths of polymorphisms are observed.

“Haplotype,” as used herein, refers to a genetic variant or combinationof variants carried on at least one chromosome in an individual. Ahaplotype often includes multiple contiguous polymorphic loci. All partsof a haplotype, as used herein, occur on the same copy of a chromosomeor haploid DNA molecule. Absent evidence to the contrary, a haplotype ispresumed to represent a combination of multiple loci that are likely tobe transmitted together during meiosis. Each human carries a pair ofhaplotypes for any given genetic locus, consisting of sequencesinherited on the homologous chromosomes from two parents. Thesehaplotypes may be identical or may represent two different geneticvariants for the given locus. Haplotyping is a process for determiningone or more haplotypes in an individual. Haplotyping may include use offamily pedigrees, molecular techniques and/or statistical inference.

A “variant” or “genetic variant” as used herein, refers to a specificisoform of a haplotype found in a population, the specific formdiffering from other forms of the same haplotype in at least one, andfrequently more than one, variant sites or nucleotides within the regionof interest in the gene. The sequences at these variant sites thatdiffer between different alleles of a gene are termed “gene sequencevariants,” “alleles,” or “variants.” The term “alternative form” refersto an allele that can be distinguished from other alleles by having atleast one, and frequently more than one, variant sites within the genesequence. “Variants” include isoforms having single nucleotidepolymorphisms (SNPs) and deletion/insertion polymorphisms (DIPs).Reference to the presence of a variant means a particular variant, i.e.,particular nucleotides at particular polymorphic sites, rather than justthe presence of any variance in the gene.

“Isoform,” as used herein, means a particular form of a gene, mRNA, cDNAor the protein encoded thereby, distinguished from other forms by itsparticular sequence and/or structure. For example, the ApoE 4 isoform ofapolipoprotein E as opposed to the ApoE 2 or ApoE 3 isoforms.

The term “genotype” in the context of this invention refers to theparticular allelic form of a gene, which can be defined by theparticular nucleotide(s) present in a nucleic acid sequence at aparticular site(s). Genotype may also indicate the pair of allelespresent at one or more polymorphic loci. For diploid organisms, such ashumans, two haplotypes make up a genotype. Genotyping is any process fordetermining a genotype of an individual, e.g., by nucleic acidamplification, DNA sequencing, antibody binding, or other chemicalanalysis (e.g., to determine the length). The resulting genotype may beunphased, meaning that the sequences found are not known to be derivedfrom one parental chromosome or the other.

“Treat,” “treating,” or “treatment” as used herein refers to any type ofmeasure that imparts a benefit to a patient afflicted with or at riskfor developing a disease, including improvement in the condition of thepatient (e.g., in one or more symptoms), delay in the onset orprogression of the disease, etc. Treatment may include any drug, drugproduct, method, procedure, lifestyle change, or other adjustmentintroduced in attempt to effect a change in a particular aspect of asubject's health (i.e., directed to a particular disease, disorder, orcondition).

“Drug” or “drug substance,” as used herein, refers to an activeingredient, such as a chemical entity or biological entity, orcombinations of chemical entities and/or biological entities, suitableto be administered to a subject to (a) delay the onset or progression ofAlzheimer's disease. In accordance with the present invention, the drugor drug substance is pioglitazone or a pharmaceutically acceptable saltthereof.

The term “drug product,” as used herein, is synonymous with the terms“medicine,” “medicament,” “therapeutic intervention,” or “pharmaceuticalproduct.” Most preferably, a drug product is approved by a governmentagency for use in accordance with the methods of the present invention.A drug product, in accordance with the present invention, contains lowdose pioglitazone.

“Disease,” “disorder,” and “condition” are commonly recognized in theart and designate the presence of signs and/or symptoms in an individualor patient that are generally recognized as abnormal and/or undesirable.Diseases or conditions may be diagnosed and categorized based onpathological changes. The disease or condition may be selected from thetypes of diseases listed in standard texts, such as Harrison'sPrinciples of Internal Medicine, 1997, or Robbins Pathologic Basis ofDisease, 1998.

“Mitochondrial dysfunction,” as used herein, means any detrimentalabnormalities of the mitochondria within a cell or cells. AD and stagesthat advance to AD are presently known in the art to be associated withmitochondrial dysfunction. This mitochondrial dysfunction causes celldamage and death by compromising ATP production, disrupting calciumhomeostasis and increasing oxidative stress. Furthermore, mitochondrialdamage can lead to apoptotic cell death by causing the release ofcytochrome c and other pro-apoptotic factors into the cytoplasm (forreview, see Wallace 1999 Science 283: 1482-1488; Schapira 2006 TheLancet 368: 70-82). Regarding a specific example found herein, and notwishing to be bound by theory, the ApoE 3 and ApoE 4 isoforms arehypothesized to cause mitochondrial dysfunction through interactionswith TOMM40. Some TOMM40 variants may act synergistically with ApoE 3isoform to accelerate mitochondrial decline. In addition, in someembodiments the ApoE 2 isoform is thought to be protective againstmitochondrial dysfunction.

As used herein, the “short” TOMM40 rs10524523 allele has less than 19thymidine (T) residues, and the “long” TOMM40 rs10524523 allele has 19or greater T residues. In some embodiments, the long allele may indicatea higher risk of onset of late onset Alzheimer's disease within a setperiod of time (e.g., over a 5-7 year period).

The rs10524523 (“523”) allele, an intronic polyT tract in the TOMM40gene, is highly polymorphic with respect to length (i.e., number of Tresidues), and variable sizes are associated with age-of-onsetdistributions of late-onset AD. Measurements of the number of T residuesat each of the 2 copies of the 523 polyT, 1 on each chromosome, that arecarried by each individual comprise the 523 genotype and can be assessedby standard procedures, such as Sanger sequencing or electrophoreticassay.

Categorical designations of each 523 polyT are assigned according tohomopolymer length: Short (S, homopolymer length less than 19 Tresidues), Long (L, length greater than or equal to 19, but shorter than30) and Very Long (VL, length greater than 29 T residues). Six different523 genotypes, using the categorical designations, are thus possible:(S,S), (VL, VL), (S,L), (VL,L), (S,VL), (L,L). See also U.S. PatentApplication Publication No. 2011/0166185 to Roses, which is incorporatedby reference herein.

APOE genotype is a well established risk factor for age of onset of AD.APOE ε4 alleles are strongly linked to the 523 long (L) allele and,therefore, individuals who have the 523 L,L genotype usually (e.g., 98%for Caucasian) possess the APOE ε4/ε4 genotype. However, the 523 short(S) and 523 very long (VL) alleles can be linked to either APOE ε2 orAPOE ε3 alleles. APOE ε2 alleles are associated with a later age ofonset of AD relative to people who carry the ε3 allele (5-8 years later,comparing APOE ε2/ε3 individuals with APOE ε3/ε3). Therefore, in someembodiments, APOE may be included in the determination in order toassign all people carrying the APOE ε2 allele to the low-risk group atthe appropriate age range. The 523 genotype provides higher resolutionfor age of onset of cognitive impairment for individuals who carry theAPOE ε3 allele in APOE (ε/3/ε3) and the APOE (ε3/ε4) genotypes.

In some embodiments, a subject with two copies of the long TOMM40rs10524523 allele is at greater risk of developing AD as compared to asubject with one copy of the long TOMM40 rs10524523 allele, or twocopies of the short TOMM40 rs10524523 allele. In some embodiments, asubject with one copy of the long TOMM40 rs10524523 allele is at greaterrisk of developing AD as compared to a subject with two copies of theshort TOMM40 rs10524523 allele. Determination of the risk of developingAD or the onset of a stage or symptom thereof based upon TOMM40 genotypeshould be performed in accordance with other risk factors such as age,and may also include APOE status in some embodiments. In someembodiments, a cognitively normal subject older than 62 years of agewith two copies of the very long TOMM40 rs10524523 allele is atdecreased risk of developing AD relative to a subject with one or twocopies of the long allele of rs10524523.

Detection of a genetic variant of TOMM40 may be performed as describedin WO 2010/019550 or US 2011/0166185, each herein incorporated byreference in its entirety.

As used herein, a “subject at risk of developing Alzheimer's disease”means one who is predisposed to Alzheimer's disease, has geneticsusceptibility for Alzheimer's disease and/or is more likely to developAlzheimer's disease at a predetermined age than subjects in which thegenetic risk factor is absent.

As used herein, “increased risk” means likely to develop AD within ashort time, e.g., 5-7 years from a time point of, for example, theinitiation of treatment according to some embodiments described herein,or the time of determination of a predisposition to or symptom ofAlzheimer's disease (for example by analysis of any one of brainatrophy, decreased synaptic activity in the brain, increased amyloidaccumulation in the brain, decreased mitochondrial function in thebrain, decreased proliferation in the brain, diseased neurons, theformation of neurofibrillar tangles in the brain, amyloid in the CSF andTau and/or phospho-Tau in the CSF).

“Increased risk” may also mean an individual is likely to develop AD ata younger age than a control subject, that is that an individual with atleast one copy of the long rs10524523 allele is at greater risk ofdeveloping AD at an earlier age than an individual with no copies of thelong rs10524523 allele according to some embodiments.

The age at which a subject is deemed to be at increased risk ofdeveloping AD may be determined by graphing one or more factors (e.g.,TOMM40 523 genotype) against age and determining the point at which therisk changes are largest related to a change in age (see FIG. 2). Thispoint may be “about” a particular age, meaning that the age may vary by0.5, 1, 2, 3, 4 or 5 years from that point, which variation may resultfrom, e.g., further optimization or higher data resolution of the graphsupon receipt of additional data.

A method of “administration” useful according to the invention includes,but is not limited to, administration by, for example, ingestion via theoral route, intranasal, rectal, inhalation, topical or injection, suchas intravenous, subcutaneous, intramuscular, intraperitoneal,intracranial and spinal injection. Additional methods of administrationare provided herein below in the section entitled “Dosage andAdministration.”

As used herein, “diagnosing” or “identifying a patient or subject havingAlzheimer's disease” refers to a process of determining if an individualis afflicted with Alzheimer's disease or a stage that progresses toAlzheimer's disease, as defined herein. A diagnosis of Alzheimer'sdisease may be based on, for example, National Institute of Neurologicaland Communicative Disorders and Stroke-Alzheimer's Disease and RelatedDisorders Association criteria.

“Low dose pioglitazone” refers to pioglitazone or a pharmaceuticallyacceptable salt thereof in an amount in the range of from 0.5 mg to 12mg, such as 0.5 mg, 0.75 mg, 1 mg, 1.25 mg, 1.5 mg, 1.75 mg, 2 mg, 2.25mg, 2.5 mg, 2.75 mg, 3 mg, 3.25 mg, 3.5 mg, 3.75 mg, 4 mg, 4.25 mg, 4.5mg, 4.75 mg, 5 mg, 5.25 mg, 5.5 mg, 5.75 mg, 6 mg, 6.25 mg, 6.5 mg, 6.75mg, 7 mg, 7.25 mg, 7.5 mg, 7.75 mg, 8 mg, 8.25 mg, 8.5 mg, 8.75 mg, 9mg, 9.25 mg, 9.5 mg, 9.75 mg, 10 mg, 10.25 mg, 10.5 mg, 10.75 mg, 11 mg,11.25 mg, 11.5 mg, 11.75 mg or 12 mg. Alternatively, in some embodimentsof the present invention, low dose pioglitazone means a low dose amountof pioglitazone or a pharmaceutically acceptable salt thereof thatprovides a pioglitazone AUC in a subject in a range of from about 0.15μg·h/mL to about 3.6 μg·h/mL (±25%). For example, low dose pioglitazoneAUC may be in a range of from 0.12, 0.37, or 1.12 to 3.4 or 4.5 μg·h/mL.

As used herein, “control subject” means a subject that has not beendiagnosed with Alzheimer's disease and/or does not exhibit anydetectable symptoms associated with Alzheimer's disease. A “controlsubject” also means a subject that is not at risk of developingAlzheimer's disease, as defined herein.

As used herein, a “subject that is not at risk of developing Alzheimer'sdisease” means, for example, a subject that does not have a TOMM40rs10524523 genotype that indicates, together with age and possibly otherfactors such as APOE status, that the subject is not more likely thanthe general population or a stratified portion thereof to develop AD ora stage or symptom thereof.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use with pioglitazone when in contact with the tissues ofsubjects, e.g., animals, including mammals, humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge, etal. describes pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 66: 1-19 (1977). The salts can be prepared insitu during the final isolation and purification of the compounds of theinvention, or separately by reacting the free base function with asuitable organic acid. Examples of pharmaceutically acceptable include,but are not limited to, nontoxic acid addition salts which are salts ofan amino group formed with inorganic acids, such as hydrochloric acid,hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, orwith organic acids, such as acetic acid, maleic acid, tartaric acid,citric acid, succinic acid or malonic acid, or by using other methodsused in the art such as ion exchange. Other pharmaceutically acceptablesalts include, but are not limited to, adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. Representative alkali or alkaline earth metal saltsinclude sodium, lithium, potassium, calcium, magnesium, and the like.Further pharmaceutically acceptable salts include, when appropriate,nontoxic ammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and arylsulfonate.

II. ALZHEIMER'S DISEASE

Symptoms of Alzheimer's Disease

Common symptoms of Alzheimer's disease include, but are not limited to,memory loss, difficulty performing familiar tasks, problems withlanguage, disorientation to time and place, poor or decreased judgment,problems with abstract thinking, misplacing things, change in mood orbehavior, changes in personality and loss of initiative. These symptomsappear gradually over time and usually (but not always) begin withepisodic memory problems, followed by other cognitive deficits thatadversely affect a person's normal functioning (i.e., activities ofdaily living). Behavioral/personality changes usually occur later in thedisease process, as a person becomes more moderately and severelyaffected. Some examples of these characteristic symptoms are describedbelow.

Memory Loss

This includes forgetting recently learned information and is one of themost common early signs of dementia. A person begins to forget moreoften and is unable to recall the information later. This includesforgetting names or appointments occasionally.

Difficulty Performing Familiar Tasks

People with dementia often find it hard to plan or complete everydaytasks. Individuals may lose track of the steps involved in preparing ameal, placing a telephone call or playing a game. This includesoccasionally forgetting why you came into a room or what you planned tosay.

Problems with Language

People with Alzheimer's disease often forget simple words or substituteunusual words, making their speech or writing hard to understand. Theymay be unable to find the toothbrush, for example, and instead ask for“the thing for my mouth.” This includes forgetting names or appointmentsoccasionally.

Disorientation to Time and Place

People with Alzheimer's disease can become lost in their ownneighborhood, forget where they are and how they got there, and not knowhow to get home. This includes forgetting the day of the week or whereyou were going. In some patients, confusion and sometimes accompanyingagitation and behavioral issues manifest more in the late afternoon orearly evening, a symptom referred to as “sundowning.”

Poor or Decreased Judgment

Those with Alzheimer's may dress inappropriately, wearing several layerson a warm day or little clothing in the cold. They may show poorjudgment, like giving away large sums of money to telemarketers. Thisincludes making a questionable or debatable decision from time to time.

Problems with Abstract Thinking

Someone with Alzheimer's disease may have unusual difficulty performingcomplex mental tasks, like forgetting what numbers are for and how theyshould be used. This includes finding it challenging to balance acheckbook.

Misplacing Things

A person with Alzheimer's disease may put things in unusual places: aniron in the freezer a wristwatch in the sugar bowl. This includesmisplacing keys or wallet temporarily.

Change in Mood or Behavior

Someone with Alzheimer's disease may show rapid mood swings—from calm totears to anger—for no apparent reason. This includes occasionallyfeeling sad or moody.

Changes in Personality

Personalities of people with dementia can change dramatically. They maybecome extremely confused, suspicious, fearful or dependent on a familymember. People's personalities do change somewhat with age.

Loss of Initiative

A person with Alzheimer's disease may become very passive, sitting infront of the TV for hours, sleeping more than usual or not wanting to dousual activities. This includes feeling weary of work or socialobligations.

Diagnosis and Staging of Alzheimer's Disease

The clinical diagnosis of Alzheimer's disease is a process thattypically involves a variety of steps (including medical history,physical and mental status examinations, and laboratory tests) andtools. Of the latter, since 1984, the diagnostic criteria established bythe National Institute of Neurological Disorders and Stroke(NINDS)/Alzheimer's Disease and related Disorders Association (ADRDA)have been, along with the DSM-IV criteria, the primary standards used inclinical practice and research. Both require the presence of memorydysfunction and cognitive impairment, although while the DSM criteriastipulate that the latter adversely affects normal functioning, theNINCDS/ADRDA criteria do not. A feature of both sets of criteria is thatthey do not consider an antemortem diagnosis of AD as definitive, sinceuntil recently there was no methodology to assess brain pathology forcharacteristic AD features until after a patient's death. TheNINCDS/ADRDA criteria therefore considered the antemortem diagnosis tobe either “possible” or “probable”, depending on the strength of theclinical evidence, including the ruling out of multiple differentialdiagnoses.

Until recently, the deterioration of a subject to Alzheimer's diseasehas been characterized by multiple clinical stages. The term “stage” isused herein in a general sense to describe how a subject's abilitieschange from normal function, e.g., normal cognitive state, toAlzheimer's disease. It should be noted that stages are general guides,symptoms can vary greatly in and/or between the stages, and that notevery subject will experience the same symptoms in a given stage orprogress to Alzheimer's disease at the same rate. For example, aseven-stage framework was developed by Barry Reisberg, M.D., clinicaldirector of the New York University School of Medicine's SilbersteinAging and Dementia Research Center, which includes: Stage 1: Noimpairment; Stage 2: Very mild decline; Stage 3: Mild decline; Stage 4:Moderate decline; Stage 5: Moderately severe decline; Stage 6: Severedecline; and Stage 7: Very severe decline. In the clinical researcharena, AD has been often defined somewhat loosely as “mild”, “moderate”,or “severe” based on scores from psychometric instruments such as theMini-Mental State Examination, where, for example, mild AD could beconsidered 18-26, moderate 11-17, and severe anything 10 or below (on a30-point scale where higher scores indicate greater cognitive function).

In 2007, Dubois et al proposed that the NINCDS/ADRDA criteria for ADdiagnosis be revised to incorporate learnings from the growth in thefield's understanding of the disease process and the development of newmethods to assess antemortem biomarkers of AD, including brain imaging(Dubois et al. 2007 Lancet Neurol 6: 734-746). In this proposal, evenwith the presence of supportive features, the antemortem diagnosis isstill considered “probable” AD, while a “definite” AD diagnosis wasreserved for histopathological confirmation or genetic evidence(mutation on chromosome 1, 14, or 21).

In 2011, a workgroup representing the National Institute onAging/Alzheimer's Association Research Roundtable proposed similarrevisions to the NINCDS/ADRDA criteria and proposed criteria toestablish a diagnosis of MCI and MCI due to AD (Albert et al. 2011Alzheimers Dement 7: 270-279; McKhann et al. 2011 Alzheimers Dement 7:263-269). This workgroup updated criteria for all cause dementia anddementia due to AD. The workgroup retained the designations of probableAD dementia, possible AD dementia, and probable or possible AD dementiawith evidence of the AD pathophysiological process. The first twodesignations were intended for use in all clinical settings, whereas thelast designation was determined to be appropriate for research purposes.The workgroup recognized that the Alzheimer's disease progression is acontinuum and that distinguishing between MCI and dementia is a clinicalassessment of whether there is significant interference with dailyactivities.

“Preclinical AD” refers to a stage at which symptoms are sufficient tomeet the currently accepted criteria of Preclinical AD (see Dubois etal., supra). Generally speaking, preclinical AD is the long,presymptomatic phase during which time the pathophysiological processesof AD are beginning. There may be very subtle cognitive symptoms yearsbefore subjects meet the clinical criteria of MCI (Sperling et al. 2011Alzheimers Dement 7: 280-292).

“Prodromal AD” refers to a stage at which symptoms meet the currentlyaccepted criteria of Prodromal AD (see Dubois et al. supra.). Inaccordance with the present invention, prodromal AD is a symptomaticpredementia stage that generally includes MCI but not dementia, and ischaracterized by symptoms not yet severe enough to meet full Alzheimer'sdisease diagnostic criteria. The Prodromal AD stage is also referred toherein as the progressive MCI stage.

III. PIOGLITAZONE

Pioglitazone is a thiazolidinedione agent having the following chemicalstructure:

Pioglitazone HCl is a potent agonist for peroxisomeproliferator-activated receptor gamma (PPARγ). PPAR receptors are foundin tissues such as adipose tissue, skeletal muscle and liver.

While not wishing to be bound by theory, it is thought that the PPARγagonist pioglitazone protects against or ameliorates at least some ofthe pathological mechanisms involved in Alzheimer's disease (AD), suchas the decrease in metabolic activity seen in the preclinical stage.

The pathophysiological changes corresponding to the clinicalmanifestation of AD may begin years, or even decades, before the firstcognitive symptoms appear, developing slowly over a preclinical phase.In some embodiments, administration of low dose pioglitazone as taughtherein may protect against or ameliorate these changes, leading to adelay in the onset cognitive impairment of the Alzheimer's type.

In some embodiments, pioglitazone is administered in an amount effectiveto protect or increase neuronal mitochondrial function, or to expand themitochondrial reservoir, for treating, such as delaying or preventing,cognitive impairment (e.g., cognitive impairment of the Alzheimer'stype). In some embodiments, treatment is initiated before significantpathological damage has accrued and/or cognitive impairment is detectedor diagnosed.

Mitochondrial dysfunction is thought to play a significant role in thecerebral hypometabolism observed in AD. Brain metabolic activity,primarily due to mitochondrial activity, decreases and non-pathologicalbrain atrophy occurs during healthy aging (Curiati et al. 2011 Am JNeuroradiol 32: 560-565), but metabolic decline and atrophy occur at asignificantly higher rate in prodromal and symptomatic early onset(Familial) AD, in mild cognitive impairment (MCI), and in late onsetAlzheimer's disease (Reiman et al. 1996 N Engl J Med 334: 752-758;Mosconi et al. 2004 Psychiatry Research: Neuroimaging 130: 141-151;Mosconi et al. 2005 J Neurol Neurosurg Psychiatry 76: 15-23; Mosconi etal. 2006 J Nucl Med 47: 1778-1786; Chételat et al. 2008 Brain 131:60-71; Mosconi et al. 2008 Annals of the New York Academy of Sciences1147: 180-195; Mosconi et al. 2009 Neurology 72: 513-520; Mosconi et al.2009 Eur J Nucl Med Mol Imaging 36: 811-822; Villain et al. 2010 Brain133: 3301-3314). Mitochondrial enzyme activity has also been found to bereduced in autopsied hippocampus of AD patients, and in platelets andfibroblasts, relative to cognitively normal subjects (Mancuso et al.2010 Adv Exp Med Biol 685: 34-44).

The hypothesis that perturbation of mitochondrial function is a veryearly event in AD etiology, occurring possibly decades ahead of clinicalsymptoms, is well-supported (Castellani et al. 2002 Journal ofNeuroscience Research 70: 357-360; Bubber et al. 2005 Annals ofNeurology 57: 695-703; Beal 2007 Mitochondrial Biology: New Perspectives287: 183-192; discussion 192-186; Liang et al. 2008 PhysiologicalGenomics 33: 240-256; Liang et al. 2008 PNAS 105: 4441-4446; Jack et al.2009 Brain 132: 1355-1365; Moreira et al. 2010 Biochimica et BiophysicaActa (BBA)—Molecular Basis of Disease 1802: 2-10; Swerdlow et al. 2010 JAlzheimers Dis 20 Suppl 2: S265-279; Cunnane et al. 2011 Nutrition 27:3-20). There are changes in expression, in multiple brain regions, ofgenes involved in mitochondrial function in young individuals who are atincreased risk of developing AD due to carriage of APOEε4(Conejero-Goldberg et al. 2011 Molecular Psychiatry 16: 836-847), andrelatively decreased metabolic activity has been measured,biochemically, following death and with imaging techniques during life,in brains of cognitively normal people who are determined to be atincreased risk of developing late onset AD because of family history ofthe disease or carriage of at least one APOEε4 allele (Small et al. 1995JAMA 273: 942-947; Reiman et al. 2005 PNAS 102: 8299-8302; Mosconi etal. 2008 Annals of the New York Academy of Sciences 1147: 180-195;Langbaum et al. 2010 Arch Neurol 67: 462-468; Mosconi et al. 2011Journal of Alzheimer's Disease).

The human brain consumes more energy per gram of tissue than any otherorgan, accounting for approximately a fifth of the body's total energyexpenditures. Glucose is the primary fuel for brain metabolism, with themajority of cellular energy production occurring in mitochondria.Neuronal mitochondria generate adenosine triphosphate (ATP) to powerneurotransmitter release and uptake at synapses, to maintain iongradients, to power mitochondrial and axonal transport. Mitochondriaalso regulate calcium homeostasis and apoptosis, while dysfunctionalmitochondria produce increased levels of toxic reactive oxygen species(Mattson et al. 2008 Neuron 60: 748-766). Some studies suggest thatneurons also utilize lactate produced by the oxidation of glucose inadjacent astrocytes (Pancani et al. 2011 Cell Calcium 50: 548-558).Lactate is ultimately reduced to pyruvate in neurons and then, likeglucose, feeds into the oxidative phosphorylation pathway inmitochondria to produce ATP.

In some embodiments, changes in brain metabolic activity uponadministering may be measured to determine the optimal dosages and/orforms of administration for pioglitazone. Brain metabolic activity maybe measured using specialized techniques known in the art, includingfunctional Magnetic Resonance Imaging (fMRI), the most commonimplementation being Blood Oxygen Level Dependent (BOLD) fMRI, and[¹⁸F]-fluorodeoxyglucose-Positron Emission Tomography (FDG-PET) (Jack etal. 2000 Neurology 55: 484-490; Whitwell et al. 2007 Brain 130:1777-1786). BOLD fMRI measures the ratio of deoxyhemoglobin tooxyhemoglobin; small increases in regional neural activity result inincreased regional demand for oxygen delivery via the cerebralvasculature, resulting in an increased fMRI signal in the area. Thus,BOLD provides an indirect, but sensitive, measure of neural activity. Aquantitative measure of glucose uptake, the cerebral metabolic rate ofglucose (CMRglu), may be calculated with FDG-PET.

Cunnane et al., reviewing a substantial body of literature on FDG-PETstudies of MCI and AD, concluded that the global cerebral metabolic rateof glucose (CMRg) is reduced by approximately 20-25% in AD patientsafter correction for brain atrophy (Cunnane et al., supra). The mostconsistent FDG-PET findings in AD are reduced CMRglu in entorhinalcortex and hippocampus—two regions that are earliest affected byAD—progressing to posterior cingulate cortex, temporoparietal areas,precuneus and prefrontal cortex as the disease advances (During et al.2011 Neurological Sciences 32: 559-569; Filippi and Agosta 2011 Journalof Alzheimers Disease 24: 455-474). Reduced cerebral glucose metabolismmay also be apparent before a diagnosis of AD, at very early stages ofcognitive decline, as well as in AD-sensitive brain regions in MCI, withthe magnitude and extent of hypometabolism worsening as cognitiondeclines (Caselli et al. 2008 Arch Neurol 65: 1231-1236; Nishi et al.2010 J Neuroimaging 20: 29-36; Chetelat et al. 2008, supra).

A longitudinal study demonstrated that, for people who progressed fromnormal cognition to a clinical diagnosis of amnestic MCI, there was acorrelation between decline in cognition and reduction in metabolism inbrain regions known to be preferentially affected by AD. This decline inthe AD-sensitive regions of the brain was not evidenced in a similargroup of people who maintained stable cognition over the study (Caselliet al. 2008, supra; Chetelat et al. 2008, supra). In addition, youngadult and middle-age individuals who are cognitively normal but at riskfor AD (e.g., with family history of AD, a carrier of APOEε4, orindividuals with presymptomatic early-onset, familial AD), have reducedglucose metabolism in brain regions sensitive to AD pathology relativeto those without these risk factors (Small, et al. 1995, supra; Reimanet al. 1996, supra; Reiman et al. 2005, supra; Mosconi et al. 2006,supra; Langbaum et al. 2010, supra; Small et al. 2000 PNAS 97:6037-6042; Reiman et al. 2004 PNAS 101: 284-289). Thus, reducedmetabolism in regions of the brain affected by AD may be one of theearliest pathophysiological changes and/or indicators of future diseasein those at risk of developing the disease, and may also be correlatedwith disease progression.

As known in the art, fMRI, using Blood Oxygen Level dependent (BOLD)contrast, can be used to visualize and measure neuronal activity duringtasks, e.g., cognitive tasks, and to visualize the resting stateactivity of the brain, including the default mode network (DMN), whichis a network of brain regions that is active during the awake restingstate but deactivated during a task (Pihlajamäki and Sperling 2008Future Neurology 3: 409-421; Huettel and Larry 2009 Encyclopedia ofNeuroscience 273-281). Neuronal activity increases metabolism andregional demand for glucose and oxygen, which stimulates blood flow tothe active regions of the brain. This is the hemodynamic response (theproduct of local cerebral blood flow, the cerebral metabolic rate ofoxygen and cerebral blood volume) that is visualized by BOLD fMRI is awidely accepted indicator of neuronal activity and reflects energyconsumption (Pihlajamäki and Sperling 2008, supra; Wise and Preston 2010Drug Discovery Today 15: 973-980; Reitz et al. 2011 Nat Rev Neurol 7:137-152).

BOLD fMRI reveals that task-evoked brain activity is compromised inthose at risk of AD, and further diminishes as AD progresses (Filippiand Agosta 2011, supra). Some of the tasks used may challenge the higherorder cognitive functions that are compromised early in the diseaseprocess, including episodic and working memory. The BOLD fMRI signalchanges earliest in the medialtemporal lobe (MTL), including thehippocampus, and connected neural networks that are required forencoding or retrieving memories (Pihlajamäki and Sperling 2008, supra).Reduced neural activity is also evident in the MTL, particularly inregions of the hippocampus, of young and old cognitively normalindividuals who are at increased risk of developing AD (Pihlajamäki andSperling 2008, supra; Filippi and Agosta 2011, supra; Wu et al. 2009 JCell Physiol 220: 58-71; Jones et al. 2011 Neurology 77: 1524-1531), andthe magnitude of the BOLD fMRI signal in the posteromedial corticalregion is associated with verbal episodic memory performance incognitively normal older subjects, and is decreased as subjects progressfrom cognitive impairment to AD dementia (Pihlajamaki et al. 2010Alzheimer Disease & Associated Disorders 24: 28-36). In addition tochanges in task-evoked brain activity in preclinical, prodromal and ADdementia, fMRI and FDG-PET studies of the brain in its resting stateindicate that the functional connectivity between specific regions ofthe brain are increasingly altered as MCI and AD progress (Reiman et al.1996, supra; Filippi and Agosta 2011, supra; Jin et al. 2012 MagneticResonance Imaging 30: 48-61). BOLD-fMRI has proven to be a particularlyuseful method for measuring functional connectivity in human brain andin brains of other species, e.g., the rat. Biswal et al. recognized asearly as 1995 that there was temporal correlation of low frequencyfluctuations of blood flow and oxygenation, measured by fMRI, in regionsof the brain that were functionally related (Biswal et al. 1995 MagnReson Med 34: 537-541). These spatio-temporally coordinated fluctuationsoccur even when the brain is not engaged in a task, i.e., when the brainis at rest, and are thought to reflect spontaneous neuronal activity orbackground brain processes (Damoiseaux et al. 2011 Neurobiology ofAging; Yamasaki et al. 2012 Neurology Research International 2012). InAD, altered functional connectivity has been noted between brain regionsor systems required for higher-order cognitive processes, including inthe DMN and the systems involved in attention (Yamasaki et al. 2012,supra). Decreased resting connectivity in the DMN in specific brainregions—e.g., between the posterior cingulate cortex and temporal cortexor hippocampus and between the subcortical region, the thalamus, and anumber of cortical regions—has been reported for AD and MCI patients(Wang et al. 2011 European Journal of Radiology). By contrast, there isincreased resting state functional connectivity in frontal regions andbetween regions of the DMN and frontal parts of the brain in AD and MCI(Wang et al. 2006 NeuroImage 31: 496-504; Zhang et al. 2009 Behav BrainRes 197: 103-108).

Heretofore the ability to predict which people are more likely todevelop these pathophysiological changes, which may lead to cognitiveimpairment, and ultimately Alzheimer's dementia, has not been feasible.The TOMM40 rs10524523 genotype along with age and possibly other factorsare useful as a prognostic biomarker to determine which subjects are atrisk for developing cognitive impairment of the Alzheimer's type andprovide the opportunity to intervene in the early phase of thisprogressive and devastating disease.

PPARγ is a ligand-activated, nuclear transcription factor that impingeson many pathways implicated in the etiology of AD (Landreth et al. 2008Neurotherapeutics 5: 481-489). Its biological actions include themodulation of inflammatory gene expression and the regulation of glucoseand lipid metabolism, both of which are abnormal in AD. PPARγ also hasdirect effects on mitochondrial function and ATP production. Manythought leaders in AD research believe that mitochondrial dysfunctionplays a significant role in the cerebral hypometabolism observed in AD.

The PPARγ receptor is activated by endogenous ligands and by a number ofpharmacological agents including drugs of the thiazolidinedione (TZD)class.

Pioglitazone is marketed for the treatment of type 2 diabetes (Actos™),and treats the insulin resistance that is the hallmark by type 2diabetes by increasing the sensitivity of tissues, particularly theliver, muscle and adipose tissue, to the effects of insulin (Olefsky2000 The Journal of Clinical Investigation 106: 467-472). T2DM andinsulin resistance are risk factors for developing AD, and diabeticpatients carrying APOEε4 are at particular risk (Irie et al. 2008 ArchNeurol 65: 89-93; Rönnemaa et al. 2008 Neurology 71: 1065-1071; Bruehlet al. 2009 Journal of Clinical and Experimental Neuropsychology 32:487-493). Brains from autopsied AD patients have markedly lower levelsof insulin, insulin receptor and IRS-1 mRNA than control brains,consistent with an insulin resistance or diabetic phenotype leading someto characterize AD as type 3 diabetes (Steen et al. 2005 J AlzheimersDis 7: 63-80. Insulin receptors are found throughout the human brain,and are at particularly high concentrations in the hypothalamus,cerebellum, and cortex, and PPARγ and its coactivator, retinoid Xreceptor (RXR), are also expressed in the brain, including in thehippocampus and cortex (Inestrosa et al. 2005 Experimental Cell Research304: 91-104; Gofflot et al. 2007 Cell 131: 405-418; Morales-Garcia etal. 2011 GLIA 59: 293-307). PPARγ receptor is expressed in astrocytesand neurons, and the level of the protein is reduced by ˜40% inpostmortem brain lysates from AD patients.

Pioglitazone improves neuronal insulin resistance (Liu et al. 2010European Journal of Pharmacology 629: 153-158), and concentrations aslow as 1 nM significantly reduce cell death due to glucose deprivation,possibly because pioglitazone affords protection from hypoglycemia byincreasing mitochondrial content and/or modulating mitochondrialstructure. The drug also increases expression of NRF1, TFAM1(transcription factors required for mitochondrial biogenesis), and UCP-2(required for mitochondrial remodeling) (Miglio et al. 2009Neurochemistry International 55: 496-504).

Beneficial effects of pioglitazone have been reported in transgenicmouse models of AD, and mouse and rat models of neurodegeneration orbrain injury. The reported beneficial effects upon treatment withpioglitazone include the reduction of brain amyloid plaque burden intransgenic mouse models of AD, improved brain glucose utilization andcerebrovascular function, reduced brain inflammation, decreasedoxidative stress, improvement of pathology-related memory and learningdeficits, and increased neurogenesis in adult animals (Heneka et al.2000 Journal of Neuroscience 20: 6862-6867; Yan et al. 2003 Journal ofNeuroscience 23: 7504-7509; Heneka et al. 2005 Brain 128: 1442-1453;Pathan et al. 2006 Life Sci 79: 2209-2216; Nicolakakis et al. 2008Journal of Neuroscience 28: 9287-9296; Kaur et al. 2009 Fundamental &Clinical Pharmacology 23: 557-566; Roberts et al. 2009 ExperimentalNeurology 216: 459-470; Glatz et al. 2010 Journal of Hypertension 28:1488-1497; Nicolakakis and Hamel 2011 J Cereb Blood Flow Metab 31:1354-1370; Morales-Garcia et al. 2011, supra; Zhang, Xu et al. 2011,supra). Pioglitazone also improved cognition and hyperinsulinemia, andimproved regional cerebral blood flow in small placebo-controlledclinical trials of diabetic patients with AD or mild cognitiveimpairment (Hanyu et al. 2009 Journal of the American Geriatrics Society57: 177-179; Hanyu et al. 2010 J Am Geriatr Soc 58: 1000-1001; Sato etal. 2010 Neurobiology of Aging 32: 1626-1633).

The marketed 15 mg, 30 mg and 45 mg dosage of pioglitazone isappropriate for dosing for type 2 diabetes and is safe and efficaciousfor the treatment of this disease. Diabetes-level doses of pioglitazonehave been used in small clinical studies of Alzheimer's disease (Hanyuet al. 2009 Journal of the American Geriatrics Society 57: 177-179;Hanyu et al. 2010 J Am Geriatr Soc 58: 1000-1001; Sato et al. 2010Neurobiology of Aging 32: 1626-1633). In addition, in a recent clinicaltrial for Alzheimer's treatment using a differentthiazolidinedione—rosiglitazone—the type 2 diabetes dosage of the drugwas used (Risner et al. 2006 Pharmacogenomics Journal 6: 246-254; Goldet al. 2010 Dementia and Geriatric Cognitive Disorders 30: 131-146).

However, it would be preferred to limit exposure to drug if the requiredpharmacodynamic effect and efficacy may be sufficiently achieved at alower dose for the intended patient population. In this manner, thefrequency of rare or uncommon adverse events may be further reduced,thereby improving the safety.

As taught herein, and as demonstrated by the BOLD study resultspresented in the Examples below, it has been surprisingly found thatdosages significantly lower than those used for the treatment of type IIdiabetes (i.e., low dose pioglitazone) result in a change in brainmetabolism and thus may be effective in the treatment of Alzheimer'sdisease, including the delay of onset of cognitive decline (e.g.,cognitive impairment of the Alzheimer type).

V. FORMULATIONS AND MODES OF ADMINISTRATION

The invention provides for a number of drug product formulations of lowdose pioglitazone useful according to the methods of the presentinvention, including but not limited to a low strength (LS) formulation,an orally disintegrating tablet (ODT) formulation, a liquid formulation,a suspension formulation, a nasal formulation, an orally immediate,modified, controlled or extended release formulation, a transdermalformulation a rectal formulation, a topical formulation or an injectableformulation.

(a) Low Strength (LS) Formulation

The invention provides for LS formulations of low dose pioglitazone, forexample as described in U.S. Ser. No. 12/452,587 and U.S. PatentPublication No. 2010/0166853, herein incorporated by reference in itsentirety). The coated preparation of the present invention comprises acore comprising a pharmaceutically acceptable organic acid with watersolubility at 20° C. of not less than 10 mg/mL and pK_(a1) (a negativecommon logarithm of the first acid dissociation constant K_(a1)) at 25°C. of not more than 5, and a coating layer comprising pioglitazone or asalt thereof.

The coated preparation of the present invention may be a singlepreparation having a core and a coating layer, or a collection ofpreparations each having a core and a coating layer. In addition, thecoated preparation of the present invention may be a capsule produced bymixing a collection of preparations each having a core and a coatinglayer with additives as necessary and filling a capsule with themixture.

Furthermore, the coated preparation of the present invention may be atablet or caplet produced by mixing a collection of preparations eachhaving a core and a coating layer with additives and compression-moldingthe mixture.

The core of the coated preparation of the present invention may consistonly of a pharmaceutically acceptable organic acid with water solubilityat 20° C. of not less than 10 mg/mL and pK_(a1) at 25° C. of not morethan 5. Alternatively, it may consist of a composition of apharmaceutically acceptable organic acid with water solubility at 20° C.of not less than 10 mg/mL and pK_(a1) at 25° C. of not more than 5 and,for example, the below-mentioned additive and the like.

The organic acid contained in the core of the coated preparation of thepresent invention is a pharmaceutically acceptable organic acid withwater solubility at 20° C. of not less than 10 mg/mL and pK_(a1) at 25°C. of not more than 5. The water solubility at 20° C. is preferably notless than 50 mg/mL, more preferably not less than 100 mg/mL. The watersolubility at 20° C. is preferably not more than 2000 mg/mL. pK_(a1) at25° C. is preferably not more than 5, more preferably not more than 4.The pK_(a1) is preferably not less than 1. Preferred is an organic acidwith water solubility at 20° C. of not less than 300 mg/mL and pK_(a1)at 25° C. of not more than 4.

Specific examples of organic acid include one or more of citric acid,tartaric acid, malic acid and ascorbic acid, and the like. The organicacid may be any of hydrate and acidic salt. In addition, the organicacid is preferably in the form of a crystal, since the mechanicalstrength and chemical stability of the core containing the crystallineorganic acid are not degraded during the production step of thepreparation of the present invention, and in view of the acidity.

In the present specification, citric acid includes citric acidmonohydrate and anhydrous citric acid.

As the organic acid, citric acid, tartaric acid and malic acid arepreferable, and citric acid (particularly anhydrous citric acid) is morepreferable as a pharmaceutical additive.

The average particle size of the organic acid is generally 100-1500 μm,preferably 300-800 μm. The average particle size is measured, forexample, using a laser diffraction particle distribution measurementapparatus (e.g., SYNPATEC HELOS-RODOS particle distribution measurementapparatus).

While the average particle size of the core varies depending on the kindof coated preparation of the present invention, it is generally 100-1500μm, preferably 300-800 μm.

The core of the coated preparation of the present invention can becovered with a coating layer comprising pioglitazone or a salt thereof.

While the content of the organic acid in the core of the coatedpreparation of the present invention varies depending on the kind oforganic acid and the like, it is generally 20-95 parts by weight,preferably 40-80 parts by weight, per 100 parts by weight of the coatedpreparation.

With regard to pioglitazone or a salt thereof used for the coatedpreparation of the present invention, examples of the salt ofpioglitazone include pharmacologically acceptable salts such as saltswith inorganic acid, salts with organic acid, salts with acidic aminoacid and the like.

Preferable examples of the salts with inorganic acid include salts withhydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid,phosphoric acid and the like.

Preferable examples of the salts with organic acid include salts withformic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalicacid, tartaric acid, maleic acid, citric acid, succinic acid, malicacid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acidand the like.

Preferable examples of the salts with acidic amino acid include saltswith aspartic acid, glutamic acid and the like.

In addition, pioglitazone may be any of anhydride or hydrates, and thepioglitazone may be further labeled with an isotope (e.g., ³H, ¹⁴C, ³⁵S,¹²⁵I) and the like.

Pioglitazone or a pharmaceutically acceptable salt thereof is preferablypioglitazone hydrochloride.

Pioglitazone or a pharmaceutically acceptable salt thereof may bediluted with a diluent and the like that are generally known in the art.

In the coated preparation of the present invention, the median particlesize of pioglitazone and a salt thereof to be used as a startingmaterial is preferably 0.5 to 50 μm.

By adopting such a median size, a coated preparation of pioglitazone ora pharmaceutically acceptable salt thereof, which has superiordissolution, can be obtained.

The above-mentioned preferable median size is applied to pioglitazone ora pharmaceutically acceptable salt thereof used as the startingmaterial. The starting material may comprise a pulverized productobtained by pulverization during the process of producing coatedpreparation, or a mixed pulverized product obtained by pulverizationtogether with an excipient (e.g., crystalline cellulose) or the like.The median size of pioglitazone or a pharmaceutically acceptable saltthereof may change beyond the above range during a production process ofthe coated preparation of the present invention, or a preservationprocess of the coated preparation after production, by coagulation ofpioglitazone or salt thereof. The pulverization is performed using apreparation forming machine such as a mortar, a jet mill, a hammer mill,a screen mill (P-3; Showa Kagaku Kikai Kosakusho Co., Ltd.) or the like.

As used herein, the median size means a particle size that divides intocrude particles and fine particles by 50% based on the weightdistribution or number distribution. The median size can be measured,for example, by laser diffraction particle size distribution measurementapparatus (e.g., SYNPATEC HELOS-RODOS particle distribution measurementapparatus).

The dispersibility of pioglitazone or a pharmaceutically acceptable saltthereof having the above-mentioned desired median size is preferably asdefined by particles of not more than 0.1 μm are contained at not morethan 10% of the total amount, and particles of not less than 1000 μm arecontained at not more than 10% of the total amount. The lower limitthereof is generally as defined by particles of not more than 0.1 μm arecontained at not less than 0.1% of the total amount, and particles ofnot less than 1000 μm are contained at not less than 0.1% of the totalamount.

While the content of pioglitazone or a pharmaceutically acceptable saltthereof in the coated preparation of the present invention variesdepending on the dosage form, dose and the like of the coatedpreparation, it is generally 0.01-30 parts by weight, preferably 0.5-25parts by weight, further preferably 0.5-20 parts by weight, per 100parts by weight of the coated preparation.

In the coated preparation of the present invention, a weight ratio ofpioglitazone and the aforementioned pharmaceutically acceptable organicacid is preferably 1:4-1:100, more preferably 1:4-1:20, more preferably1:5-1:10. The weight of the pioglitazone means pioglitazone equivalentin a pharmaceutically acceptable salt of pioglitazone.

In the coated preparation of the present invention, the amount of thecoating layer comprising pioglitazone or a salt thereof to be used isgenerally 5-205 parts by weight, preferably 10-100 parts by weight, morepreferably 20-90 parts by weight, per 100 parts by weight of the core.

The coated preparation of the present invention preferably containscellulose or a cellulose derivative in a coating layer. Of these, acellulose derivative is preferable.

The cellulose derivative is a cellulose wherein a part of the cellulosemolecule is substituted by other atoms or functional groups. Examples ofthe cellulose derivative include low-substituted hydroxypropylcellulose(L-HPC), hydroxypropylmethylcellulose, methylcellulose,hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcelluloseacetate succinate and the like. Of these, low-substitutedhydroxypropylcellulose is preferable. More preferred is low-substitutedhydroxypropylcellulose having a hydroxypropoxyl group content of 5-16 wt% (e.g., LH-11, LH-21, LH-31, LH-22, LH-32, LH-20, LH-30, LH-33 (tradenames, manufactured by Shin-Etsu Chemical Co., Ltd.) etc.) and the like.

The content of the cellulose or cellulose derivative in the coatinglayer of the coated preparation of the present invention is generally0.5-70 parts by weight, preferably about 2-about 50 parts by weight,more preferably about 2-about 30 parts by weight, per 100 parts byweight of the coating layer.

Since cellulose or a cellulose derivative (preferably cellulosederivative) is contained in the coating layer, the coated preparation ofthe present invention has a construct constituting a coating layer,which comprises cellulose or a cellulose derivative as a skeleton and ismaintained in an aqueous solvent, wherein pioglitazone or apharmaceutically acceptable salt thereof is dissolved in an organic acid(solution) in the construct to afford an aqueous solution. As a result,the coated preparation of the present invention can, as compared toconventional preparations, remarkably increase the maximum bloodconcentration and AUC of pioglitazone after administration, andremarkably decrease inter-individual relative standard deviation (RSD)in AUC.

In addition, since the coated preparation of the present invention has aconstruct constituting a coating layer, which comprises cellulose or acellulose derivative as a skeleton and is maintained in an aqueoussolvent, wherein pioglitazone or a pharmaceutically acceptable saltthereof is dissolved in an organic acid (solution) in the construct toafford an aqueous solution, it can enhance bioavailability as comparedto conventional preparations. Specifically, the bioavailability of thecoated preparation of the present invention exceeds 75% when thepreparation is administered to dogs.

In the present specification, the bioavailability can be determined by,for example, dividing AUC at the time of non-intravenous administrationof a given amount of pioglitazone by AUC at the time of intravenousadministration of the same amount of pioglitazone. For example, when thebioavailability of a low dose pioglitazone immediate release drugproduct of the present invention is administered orally is to becalculated, the formula may be as the following:

Bioavailability(%)=(AUC of oral administration/AUC of intravenousadministration)×100.

When pioglitazone is dissolved in the construct to afford an aqueoussolution, a similar effect as achieved by the administration of solutioncan be provided, which is expected to increase maximum bloodconcentration, AUC and bioavailability.

Here, the aqueous solvent in the present specification includes water,KCl—HCl buffer (e.g., KCl—HCl buffer at pH 2.0), Mcllvaine buffer (e.g.,Mcllvaine buffer at pH 2.2, pH 2.5 or pH 3.0) and the like. Theconstruct constituting a coating layer, which comprises a cellulosederivative as a skeleton and is maintained in an aqueous solventspecifically means, for example, that the construct is present for notless than 10 minutes preferably in KCl—HCl buffer (pH 2.0, 900 mL) underconditions of Paddle Method (50 rpm), more preferably in Mcllvainebuffer (pH 2.2, 900 ml) under conditions of Paddle Method (50 rpm),still more preferably in Mcllvaine buffer (pH 2.5, 900 ml) underconditions of Paddle Method (50 rpm), particularly preferably inMcllvaine buffer (pH 3.0, 900 mL) under conditions of Paddle Method (50rpm).

The Paddle Method in the present specification means measurementaccording to the Japanese Pharmacopoeia 14th Edition, General Tests,Dissolution Test Method 2, unless particularly indicated.

The coated preparation of the present invention may contain additivesconventionally used in the technical field of formulation ofpreparations. Examples of the additive include excipient, disintegrant,binder, lubricant, colorant, pH regulator, surfactant, stabilizer,corrigent, sweetener, flavor, glidant, antistatic agent, light shieldingagent, antioxidant, reducing agent, chelating agent and the like. Theseadditives are used in an amount conventionally employed in the technicalfield of formulation of preparations. In addition, these additives maybe used in a mixture of two or more kinds thereof in an appropriateratio.

Examples of the excipient include saccharides; crystalline cellulose;starches such as corn starch, potato starch, wheat starch, rice starch,partly pregelatinized starch, pregelatinized starch, porous starch,dextrin, carboxymethyl starch and the like; anhydrous calcium phosphate,precipitated calcium carbonate, calcium silicate, powder cellulose,gelatin, light anhydrous silicic acid, synthetic aluminum silicate,magnesium aluminometasilicate, magnesium oxide, calcium phosphate,calcium carbonate, calcium sulfate.

Examples of saccharides include sugar, starch sugar, lactose, honey andsugar alcohol. Two or more kinds of these saccharides may be used in amixture in an appropriate ratio.

Examples of sugar include sucrose, white soft sugar, glycosyl sucrose[coupling sugar (trade name)], fructooligosaccharide and palatinose.

Examples of starch sugar include glucose, maltose, powdered starchsyrup, starch syrup, fructose and trehalose.

Examples of lactose include lactose, isomerized lactose (lactulose) andhydrogenated lactose (lactitol).

Examples of honey include various kinds of honey generally used foreating.

Examples of sugar alcohol include sorbitol, mannitol (specifically,D-mannitol), maltitol, hydrogenated glucose syrup, xylitol, reducedparatinose and erythritol.

The saccharides are preferably sugar alcohol, starch sugar and sucrose,more preferably mannitol, trehalose and sucrose. Of these, mannitol andtrehalose are preferable. From the aspect of suppressing color change ofthe preparation (specifically color change under preservationconditions), in the coated preparation of the present invention, thecoating layer is preferably to contain mannitol or trehalose.

When saccharides are used for the coated preparation, the contentthereof is for example, 5-90 parts by weight, preferably 5-40 parts byweight, per 100 parts by weight of the coated preparation.

Particularly, when the coated preparation of the present inventioncontains mannitol or trehalose, the content of mannitol or trehalose ispreferably 5-40 parts by weight, more preferably 5-30 parts by weight,per 100 parts by weight of the coated preparation.

Examples of crystalline cellulose include CEOLUS KG801, KG802, PH101,PH102, PH301, PH302, PH-F20, RC-A591NF (trade names, manufactured byAsahi Kasei Chemicals Corporation), including one calledmicrocrystalline cellulose.

Examples of disintegrants include carboxymethylcellulose, calciumcarboxymethylcellulose (carmellose calcium), sodium carboxymethylstarch, carmellose sodium, croscarmellose sodium, crospovidone[preferably, Kollidon CL, CL-M, CL-F, CL-SF (trade name, BASF JAPANLTD.); Polyplasdone XL, XL-10, INF-10 (trade name, ISP JAPAN LTD.)],low-substituted hydroxypropylcellulose [preferably low-substitutedhydroxypropylcellulose having a hydroxypropoxyl group content of 5-16 wt%, such as LH-11, LH-21, LH-31, LH-22, LH-32, LH-20, LH-30, LH-33 (tradename, manufactured by Shin-Etsu Chemical Co., Ltd.) etc.], hydroxypropylstarch, cornstarch and partly pregelatinized starch.

When a disintegrant is used for the coated preparation of the presentinvention, the content of the disintegrant is, for example, 0.5-50 partsby weight, preferably 1-25 parts by weight, per 100 parts by weight ofthe coated preparation.

Examples of binders include hydroxypropylcellulose [preferably HPC-SSL,SL, L (trade name, NIPPON SODA CO., LTD.)],hydroxypropylmethylcellulose, povidone (polyvinylpyrrolidone), arabicgum powder, sucrose, gelatin, pullulan, methylcellulose, crystallinecellulose, low-substituted hydroxypropylcellulose [preferablylow-substituted hydroxypropylcellulose having a hydroxypropoxyl groupcontent of 5-16 wt %, such as LH-11, LH-21, LH-31, LH-22, LH-32, LH-20,LH-30, LH-33 (trade name, manufactured by Shin-Etsu Chemical Co., Ltd.)etc.], macrogol, dextran, polyvinyl alcohol and starch paste. Of these,hydroxypropylcellulose is preferable.

When a binder is used for the coated preparation of the presentinvention, the content of the binder is, for example, 0.01-50 parts byweight, preferably 0.1-10 parts by weight, per 100 parts by weight ofthe coated preparation.

Examples of lubricants include stearic acid, magnesium stearate, calciumstearate, talc, sucrose esters of fatty acids, sodium stearyl fumarate,waxes, DL-leucine, sodium lauryl sulfate, magnesium lauryl sulfate,macrogol and light anhydrous silicic acid (e.g., AEROSIL). Of these,magnesium stearate is preferable.

Examples of colorants include food colors such as Food Yellow No. 5(Sunset Yellow, same as Food yellow No. 6 in the US), Food Red No. 2,Food Blue No. 2 and the like, food lake colors, yellow ferric oxide(yellow iron oxide), diiron trioxide (red iron oxide), riboflavin,riboflavin organic acid ester (e.g., riboflavin butyrate), riboflavinphosphate or alkali metal salt thereof or alkaline earth metal saltthereof, phenolphthalein, titanium oxide, lycopene, beta-carotene.

Examples of the pH regulator include citrate, phosphate, carbonate,tartrate, fumarate, acetate and amino acid salt.

Examples of the surfactant include sodium lauryl sulfate, polysorbate80, polyoxyethylene (160) polyoxypropylene (30) glycol, polyoxyethylene(196) polyoxypropylene (67) glycol and polyoxyethylene hydrogenatedcastor oil 60.

Examples of the stabilizer include sodium ascorbate, tocopherol,tetrasodium edetate, nicotinamide, cyclodextrins; alkaline earth metalsalts (e.g., calcium carbonate, calcium hydroxide, magnesium carbonate,magnesium hydroxide, magnesium silicate, magnesium aluminate) andbutylhydroxyanisole.

Examples of the corrigent include ascorbic acid, (anhydrous) citricacid, tartaric acid and malic acid.

Examples of the sweetener include aspartame, acesulfame potassium,thaumatin, saccharin sodium and dipotassium glycyrrhizinate. Of these,aspartame is preferable.

Examples of the flavor include menthol, peppermint oil, lemon oil andvanillin.

Examples of the glidant include light anhydrous silicic acid andhydrated silicon dioxide. Here, the light anhydrous silicic acid may beany containing hydrated silicon dioxide (SiO₂ nH₂O) (n is an integer) asa main component and, as concrete examples thereof, Sylysia 320 (tradename, FUJI SILYSIA CHEMICAL LTD.), AEROSIL 200 (trade name, NIPPONAEROSIL CO., LTD.) and the like can be used.

Examples of the antistatic agent include talc and light anhydroussilicic acid.

Examples of the light shielding agent include titanium oxide.

Examples of the antioxidant include dibutylhydroxytoluene (BHT),tocopherol, tocopherol ester (e.g., tocopherol acetate), ascorbic acidor alkali metal salt thereof or alkaline earth metal salt thereof,lycopene, beta-carotene.

Examples of the reducing agent include cystine and cysteine.

Examples of the chelating agent include EDTA or alkali metal saltthereof or alkaline earth metal salt thereof.

The coated preparation of the present invention may have an intermediatelayer formed between the core and the coating layer comprisingpioglitazone or a salt thereof. Using such intermediate layer, anadverse effect (e.g., decomposition of pioglitazone) of the organic acidin the core on pioglitazone or a salt thereof in the coating layer canbe prevented, and the durability of the coated preparation can beprolonged.

The dosage form of the coated preparation of the present invention isgenerally a solid preparation. Examples of the solid preparation includetablet, caplet, capsule, powder, granule and troche. Of these, granule,capsule and tablet are preferable. Semi-solid dosage forms, such as agel containing the coated preparation, and liquid preparationscontaining a solution of pioglitazone of the appropriate dosage are alsouseable in accordance with the present invention.

The shape of the solid preparation is not particularly limited, and maybe any of round, caplet, doughnut, oblong and the like.

The solid preparation may be coated with a coating agent, and may have amark and letters for identification and further a score line forpartition.

Examples of the coating base include sugar coating base, aqueous filmcoating base, enteric film coating base, sustained-release film coatingbase and the like.

As the sugar coating base, sucrose is used and one or more kindsselected from talc, precipitated calcium carbonate, gelatin, gum arabic,pullulan, carnauba wax and the like may be used in combination.

Examples of the aqueous film coating base include cellulose polymerssuch as hydroxypropylcellulose, hydroxypropylmethylcellulose,hydroxyethylcellulose, methylhydroxyethylcellulose and the like;synthetic polymers such as polyvinylacetal diethylaminoacetate,aminoalkyl methacrylate copolymer E [Eudragit E (trade name)],polyvinylpyrrolidone and the like; polysaccharides such as pullulan andthe like; and the like.

Examples of the enteric film coating base include cellulose polymerssuch as hydroxypropylmethylcellulose phthalate,hydroxypropylmethylcellulose acetate succinate,carboxymethylethylcellulose, cellulose acetate phthalate and the like;acrylic acid polymers such as methacrylic acid copolymer L [Eudragit L(trade name)], methacrylic acid copolymer LD [Eudragit L-30D55 (tradename)], methacrylic acid copolymer S [Eudragit S (trade name)] and thelike; naturally occurring substances such as shellac and the like; andthe like.

Examples of the sustained-release film coating base include cellulosepolymers such as ethylcellulose, cellulose acetate and the like; acrylicacid polymers such as aminoalkyl methacrylate copolymer RS [Eudragit RS(trade name)], ethyl acrylate-methyl methacrylate copolymer suspension[Eudragit NE (trade name)] and the like; and the like.

Two or more kinds of the above-mentioned coating bases may be used in amixture in an appropriate ratio. In addition, coating additives may alsobe used during coating.

Examples of the coating additive include light shielding agents and/orcolorants such as titanium oxide, talc, ferric oxide and the like;plasticizers such as polyethylene glycol, triethyl citrate, castor oil,polysorbates and the like; and the like.

The coated preparation of the present invention can be produced by usingthe above-mentioned various additives according to a conventional methodin the technical field of formulation of preparations.

For example, the coated preparation of the present invention can beproduced by:

(1) mixing an organic acid with additives where necessary to give a corecontaining an organic acid,

(2) forming a coating layer comprising pioglitazone or a salt thereof onthe surface of the core by coating the core containing an organic acidwith pioglitazone or a salt thereof and additives where necessary, and

(3) drying and sieving the obtained coated product as necessary.

In addition, the coated preparation of the present invention can also beproduced by mixing the coated product after drying and sieving with anadditive as necessary, and compression molding or filling the mixture ina capsule.

Here, the mixing (including granulation, drying, milling and the like)is performed, for example, using a preparation forming machine such as aV-type mixer, a tumbler mixer, a high speed agitating granulator(FM-VG-10; POWREX CORPORATION), an all-round kneader (Hata Tekkosho,Co., Ltd.), a fluidized-bed dryer/granulator (LAB-1, FD-3S, FD-3SN;POWREX CORPORATION), a box vacuum dryer (Kusunoki Machinery Co., Ltd.),a screen mill (P-3; Showa Kagaku Kikai Kosakusho Co., Ltd.), centrifugalfluidized-bed granulator (CF-mini, CF-260, CF-360; Freund Corporation),dry granulator, spray drying granulator, rotating fluidized-bedgranulator (MP10; POWREX CORPORATION) and the like.

For coating, for example, a preparation producing machine such as acentrifugal fluidized-bed granulator (CF-mini, CF-260, CF-360; FreundCorporation), a rolling granulator (MP10; POWREX CORPORATION), a generalfluidized-bed coating apparatus, a wurster type coating apparatus andthe like is used, and a centrifugal fluidized-bed granulator ispreferably used.

The compression molding is performed, for example, by punching generallyat a pressure of 0.3-35 kN/cm² using a single-punch tableting machine(KIKUSUI SEISAKUSHO LTD.), a rotary tableting machine (KIKUSUISEISAKUSHO LTD.), Auto-graph (Shimadzu Corporation) and the like.

Examples of capsules which can be used for capsule filling includegelatin capsules (e.g., LICAPS®, CONI-SNAP® caps, PRESS-FIT® caps andXPRESS-FIT™ caps), hydroxypropylmethylcellulose (HPMC) capsules (e.g.VCAPS®), pullulan capsules and the like (preferably,hydroxypropylmethylcellulose (HPMC) capsules).

The above-mentioned core containing organic acid is coated by thefollowing method or a method analogous thereto:

1) a method including spraying pioglitazone or a salt thereof togetherwith additives as necessary (preferably, an excipient [preferablycrystalline cellulose (which may be omitted), saccharides (preferablymannitol, trehalose, sucrose)], a disintegrant (preferably L-HPC)) ontothe core containing an organic acid, while spraying a solution of abinder (preferably, hydroxypropylcellulose) in a solvent [e.g., one ormore kinds selected from water, alcohol (e.g., methanol, ethanol,propanol, isopropanol), acetone and acetonitrile; preferably water orisopropanol] (the solution may be a dispersion);

2) a method including spraying a solution of a binder (preferably,hydroxypropylcellulose) containing pioglitazone or a salt thereof, andan additive as necessary (preferably, excipient [preferably crystallinecellulose (which may be omitted), saccharides (preferably, mannitol,trehalose, sucrose)], a disintegrant (preferably, L-HPC)) in a solvent[e.g., one or more kinds selected from water, alcohol (e.g., methanol,ethanol, propanol, isopropanol), acetone, acetonitrile; preferably wateror isopropanol] (the solution may be dispersion) onto the corecontaining organic acid;

3) a method including spraying pioglitazone or a salt thereof togetherwith an additive as necessary (preferably, excipient [preferably,crystalline cellulose (which may be omitted), saccharides (preferably,mannitol, trehalose, sucrose)], a disintegrant (preferably, L-HPC), anda binder (preferably, hydroxypropylcellulose)) onto the core containingorganic acid, while, e.g., methanol, ethanol, propanol, isopropanol),acetone, acetonitrile; preferably water or isopropanol]; or

4) a method including spraying pioglitazone or a salt thereof togetherwith cellulose or a cellulose derivative [preferably, cellulosederivative (more preferably L-HPC)], and an additive as necessary(preferably, excipient [preferably crystalline cellulose (which may beomitted), saccharides (preferably, mannitol, trehalose, sucrose)] ontothe core containing organic acid, while spraying a solution of a binder(preferably, hydroxypropylcellulose) in a solvent [e.g., one or morekinds selected from water, alcohol (e.g., methanol, ethanol, propanol,isopropanol), acetone and acetonitrile; preferably water or isopropanol](the solution may be dispersion).

The core of the coated preparation of the present invention preferablyconsists of at least one kind of organic acid selected from citric acid,tartaric acid, malic acid and ascorbic acid [preferably citric acid(particularly anhydrous citric acid)].

In addition, the coating layer comprising pioglitazone or a salt thereofin the coated preparation of the present invention preferably consistsof pioglitazone or a salt thereof (preferably pioglitazonehydrochloride), an excipient [preferably crystalline cellulose (whichmay be omitted), saccharides (preferably mannitol, trehalose, sucrose;more preferably mannitol)], a disintegrant (preferably L-HPC) and abinder (preferably hydroxypropylcellulose), or it is a coating layerconsisting of pioglitazone or a salt thereof (preferably pioglitazonehydrochloride), an excipient [preferably crystalline cellulose (whichmay be omitted), saccharides (preferably mannitol, trehalose, sucrose;more preferably mannitol)], cellulose or a cellulose derivative(preferably a cellulose derivative, more preferably L-HPC) and a binder(preferably hydroxypropylcellulose).

(b) Orally Disintegrating Tablet (ODT) Formulation

The invention provides for an orally disintegrating tablet wherein theactive ingredient is pioglitazone or a pharmaceutically acceptable saltthereof (for example as described in U.S. Ser. No. 12/810,779,corresponding to US 2010-0278390, incorporated by reference in itsentirety).

Using the production method of the present invention, an orallydisintegrating tablet, which is rapidly disintegrated in an oral cavity,has desired appropriate hardness, and is superior in the storagestability since it shows only a small decrease in the hardness and asmall increase in the tablet thickness even under high temperatureand/or high humidity conditions without any packages, can be easilyproduced by simple steps. In addition, using the production method ofthe present invention, tableting troubles during tableting, such ascapping and binding to a die inner wall and the like can be suppressed.

As used herein, an orally disintegrating tablet or ODT means a tabletthat is rapidly disintegrated by saliva in an oral cavity.

The orally disintegrating tablet of the present invention may comprise(a) one or more saccharides or sugar alcohols selected from the groupconsisting of mannitol (particularly, D-mannitol), lactose(particularly, lactose hydrate), xylitol, sucrose, erythritol andglucose (to be also referred to as component (a) in the presentspecification) and (b) low substituted hydroxypropylcellulose (to bealso referred to as component (b) in the present specification).

As component (a), mannitol and lactose are preferable.

The content of component (a) is preferably 50-95 wt %, more preferably70-90 wt %, of the weight of the preparation. Component (a) can also beoptionally dissolved in water and the like as mentioned below and usedas a binding solution for agitation granulation. The content of theabove-mentioned component (a) also includes the amount used as thebinding solution. When used as the binding solution, the amount thereofis preferably less than 10 wt %, more preferably about 2-5 wt %, of thecontent of the above-mentioned component (a).

The average particle size of the saccharides and sugar alcohols ofcomponent (a) is preferably not more than 50 μm, more preferably 10-20μm. When the average particle size exceeds 50 μm, the disintegrationtime tends to be extended.

The average particle size of the saccharides and sugar alcohols of theabove-mentioned component (a) means their initial average particle sizeof the starting materials before being subjected to the agitationgranulation and means that they have a particle size within theabove-mentioned range, and the average particle size may change duringthe subsequent production processes and storage of the preparation.

The saccharides and sugar alcohols of component (a) having an averageparticle size within the above-mentioned range are commerciallyavailable. Alternatively, the commercially available products may bepulverized with a conventional method to adjust the particle size andthereafter used.

In one embodiment, the average particle size in the presentspecification shows a 50% accumulated particle size in the particle sizedistribution measured based on a dry method using an airflow-typedisperser.

In the present invention, the low substituted hydroxypropylcellulosedoes not require a particular limitation on the grade and the like, anda commercially available product can be used. For example, lowsubstituted hydroxypropylcellulose having a hydroxypropoxyl groupcontent of about 7.0-12.9 wt % can be used.

The content of the low substituted hydroxypropylcellulose is preferably3-20 wt %, more preferably 5-15 wt %, of the weight of the preparation.

The orally disintegrating tablet of the present invention preferablycontains (c) one or more saccharides or sugar alcohols selected from thegroup consisting of powder hydrogenated maltose starch syrup, maltose,maltitol, sorbitol and trehalose (to be also referred to as component(c) in the present specification). The presence of component (c) furtherincreases the tablet hardness.

As component (c), powder hydrogenated maltose starch syrup and maltoseare preferable.

The content of component (c) is preferably 0.1-5 wt %, more preferably0.1-1 wt %, of the weight of the preparation.

The orally disintegrating tablet of the present invention does notsubstantially contain a starch disintegrant (e.g., corn starch, sodiumcarboxymethyl starch, rice starch, wheat starch, pregelatinized starch,partly pregelatinized starch etc.).

Here, substantially free of in the present specification means absenceof an amount that adversely influences the storage stability ofpreparations. Specifically, the content of the starch disintegrant ispreferably not more than 5 wt %, more preferably not more than 3 wt %,still more preferably not more than 1 wt %, of the weight of thepreparation.

The orally disintegrating tablet of the present invention preferablycontains thaumatin. The content of thaumatin is preferably 0.1-5 wt %,more preferably 0.1-1 wt %, of the weight of the preparation. Thaumatinis a sweetener generally added for masking the bitterness of an activeingredient. In the present invention, the presence of thaumatin provideseffects of improved moldability during production and increasedhardness.

Besides the above-mentioned components, the orally disintegrating tabletof the present invention may contain additives generally used for solidpreparations. The additive is, for example, excipient, disintegrantother than starch disintegrant, binder, lubricant, fluidizer, corrigent,sweetening agent, coating agent, colorant, flavor and the like. Thecontent of these additives is not particularly limited and may beappropriately selected from an amount conventionally used in thepharmaceutical field. The total amount of the additives except forcomponents (a) and (b) (when component (c) is contained, the totalamount of the additives except for components (a)-(c)) is preferably notmore than 50 wt %, more preferably not more than 25 wt %, of the weightof the preparation.

The orally disintegrating tablet of the present invention containspioglitazone as an active ingredient. The content of the activeingredient may be appropriately determined based on the amount used forclinical application, and it is preferably not more than 50 wt %, morepreferably not more than 25 wt %, of the weight of the preparation.

The orally disintegrating tablet of the present invention ischaracterized by production including steps of granulating a compositioncontaining the above-mentioned components (a) and (b) (preferably theabove-mentioned components (a), (b) and (c)) by an agitation granulationmethod, and compression-molding the obtained granulation product. It isconsidered that since the granulation product becomes spherical byagitation granulation, tableting troubles (particularly, binding to dieinner wall) in the subsequent compression-molding step are prevented inthe present invention.

The production method of the orally disintegrating tablet of the presentinvention is explained in detail in the following.

1. Granulation Step

The above-mentioned components (a) and (b) (preferably theabove-mentioned components (a), (b) and (c)), an optional activeingredient and/or an optional additive are mixed. The additive is, forexample, excipients (e.g., talc), disintegrants other than starchdisintegrants (e.g., crospovidone), sweetening agents, colorants,flavors and the like. The active ingredient may be mixed with anexcipient (e.g., talc) first and then coated with a coating agent (e.g.,aqueous ethylcellulose dispersion, triacetine) for the purpose ofmasking bitterness and the like.

The above-mentioned mixture is granulated by an agitation granulationmethod. The agitation granulation method is also generally referred toas a high-speed agitation granulation method. Here, the (high-speed)agitation granulation method is a method including adding dropwise orspraying a binder solution on a mixed powder by rotating the main wingsset on the bottom of a granulating machine to form large particles, andgrinding the particles by a chopper on the side wall to give granulesdesired particle size (Yoshihisa SAGAWA, Pharmaceutical ProductPreparation Technique, CMC Publishing CO., LTD., published in 2002, page108).

The granulation by an agitation granulation method can be performed byusing what is called an agitation granulator (also referred to as ahigh-speed agitation granulator) (e.g., high-speed mixer, LFS-GS-2J(manufactured by Fukae Powtec); VERTICAL GRANULATOR (manufactured byPOWREX CORPORATION); NEW SPEED KNEADER (manufactured by OKADA SEIKO CO.,LTD.) etc.). The rotation speed of the main wings and chopper is notparticularly limited, and may be appropriately selected from the rangegenerally used at agitation granulation. Specifically, a bindingsolution (e.g., water or, where necessary, other additives may beblended) is added to the above-mentioned mixture in the agitationgranulator, and the mixture is granulated. When thaumatin is added inthe present invention, though not particularly limited, it may be addedto the binding solution.

2. Compression Molding Step

To the granulation product obtained in the granulation step is added anoptional active ingredient and/or an optional additive (e.g., fluidizers(e.g., light anhydrous silicic acid), lubricants (e.g., magnesiumstearate, sodium stearyl fumarate, calcium stearate), flavors), and themixture is blended and compression-molded by a tableting machine and thelike. The compression molding pressure (tableting pressure) may beappropriately selected from the range generally used at tabletproduction. While the pressure is not particularly limited, it ispreferably not less than 200 kg.

The orally disintegrating tablet of the present invention produced asmentioned above has desired appropriate hardness, is rapidlydisintegrated in an oral cavity, and shows superior storage stability,even though it can be easily produced without cumbersome steps ofhumidification and drying after tableting and a special facility of anexternal lubrication system.

The hardness of the orally disintegrating tablet of the presentinvention is generally about 3-6 kg when the tablet has a diameter of6-7 mm and a thickness of about 3 mm. Here, the hardness of the tabletin the present specification is a value measured by a Schleuniger tablethardness tester (Dr. Schleuniger Pharmatron AG).

While the disintegration time of the orally disintegrating tablet of thepresent invention in an oral cavity varies depending on the form ofpreparation, dose and the like, it is generally within 60 sec,preferably within 30 sec.

The orally disintegrating tablet of the present invention is notparticularly limited as regards the size and form, and may be a scoredtablet having a cleavage line.

The orally disintegrating tablet of the present invention can beingested without water.

VI. USES

The methods of the invention are used to delay onset of Alzheimer'sdisease or a phase or stage indicative of or associated with developmentof Alzheimer's disease in a patient at risk of developing Alzheimer'sdisease. The invention also provides for pharmaceuticals that can beused to delay onset of Alzheimer's disease, a symptom thereof, or aphase or stage indicative of or associated with development ofAlzheimer's disease in a patient at risk of developing Alzheimer'sdisease.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

EXAMPLES

Having now generally described the invention, the same will be morereadily understood through reference to the following Examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.

The following examples are put forth for illustrative purposes only andare not intended to limit the scope of what the inventors regard astheir invention.

Example 1 Low Dose Pioglitazone Granules 1

Pioglitazone HCl (228.1 g), mannitol (ROQUETTE, 335.8 g) and L-HPC(LH-32 Shin-Etsu Chemical Co., Ltd., 115.0 g) are mixed to give adusting powder. Hydroxypropylcellulose (HPC-SSL, NIPPON SODA CO., LTD.,9.2 g) is dissolved in purified water (194.6 g) to give a bindingliquid. Anhydrous citric acid crystal (Jungbunzlauer, 1380 g) is fedinto a centrifugal fluidized-bed granulator (CF-360, Freund Corporation)and coated with the dusting powder while spraying the binding liquid.The resulting granules are dried under reduced pressure at 40° C. for 18hr, and sieves of 16 mesh and 42 mesh are used to give granules at therange of 16-42 mesh (aperture 0.355-1.00 mm). The granules (7193.6 g)are mixed with talc (Matsumurasangyo Co., Ltd., 3.2 g) and lightanhydrous silicic acid (AEROSIL, NIPPON AEROSIL, 3.2 g) in a tumblermixer (60 L, Showa Kagaku Kikai Kosakusho Co., Ltd.) to givepioglitazone hydrochloride granules having the following composition per450 mg.

anhydrous citric acid crystal   300 mg Pioglitazone HCl 49.59 mgmannitol 73.01 mg L-HPC   25 mg hydroxypropylcellulose    2 mg talc  0.2mg light anhydrous silicic acid  0.2 mg total   450 mg

The resulting composition can be diluted in an appropriate excipient togive the desired dosage, including any of the dosages recited herein,for example, 0.5 mg, 1.5 mg, 4.5 mg and 9.0 mg. The desired dosages canthen be formulated into oral dosage forms, such as capsules, tablets orcaplets.

Example 2 Low Dose Pioglitazone Granules 2

Pioglitazone HCl (9.90 g), mannitol (ROQUETTE, 186.2 g) and L-HPC (LH-32Shin-Etsu Chemical Co., Ltd., 39.96 g) are mixed to give a dustingpowder. Hydroxypropylcellulose (HPC-SSL, NIPPON SODA CO., LTD., 12.00 g)is dissolved in purified water (340.2 g) to give a binding liquid.Anhydrous citric acid crystal (Jungbunzlauer, 400.0 g) is fed into acentrifugal fluidized-bed granulator (CF-260, Freund Corporation) andcoated with the dusting powder while spraying the binding liquid. Theresulting granules are dried under reduced pressure at 40° C. for 18hrs, and sieves of 16 mesh and 42 mesh are used to give granules ofpioglitazone hydrochloride at the range of 16-42 mesh (aperture0.355-1.00 mm) having the following composition.

anhydrous citric acid crystal 53.33 mg pioglitazone HCl 1.102 mgmannitol 20.69 mg L-HPC  4.44 mg hydroxypropylcellulose  0.36 mg total79.92 mg

Example 3 Low Dose Pioglitazone Capsules

The low dose pioglitazone granules 2 formulated in Example 2 (39.96 g)are mixed with talc (Matsumurasangyo Co., Ltd., 0.02 g) and lightanhydrous silicic acid (AEROSIL, NIPPON AEROSIL, 0.02 g) in a glassbottle to give pioglitazone hydrochloride granules having the followingcomposition per 80 mg. The pioglitazone hydrochloride granules (80 mg)are filled in No. 4 hypromellose capsules (Qualicaps Co., Ltd.) to givecapsules having the following composition.

Component amount added granule obtained in Example 2 79.92 mg talc 0.04mg light anhydrous silicic acid 0.04 mg No. 4 hypromellose capsule 1capsule

Example 3 Pioglitazone Liquid Formulation 1

A liquid formulation of pioglitazone is prepared using the materials asfollows.

Materials:

Citric Acid, Sigma, C1857, lot 089K0057

Distilled Water, Ice Mountain

HPMC, USP, Sigma, H-3785, lot 122K0149

Pioglitazone HCl, Takeda, lot 345

Polyethylene Glycol 200, Sigma, P3015, lot 098K0056

Polysorbate 80, NF, Spectrum, P0138, lot XV0879

Propylene Glycol, USP/FCC, Fisher, P355, lot 080676

Sucrose, USP, Sigma, S3929, lot 086K0022

Syrup NF, Spectrum, SY105, lot XP0703

Approximately 0.01496 g of pioglitazone HCl is transferred into a 50-mLgraduated cylinder. 0.69 g of polyethylene glycol 200 is added and mixedto wet the solids. 1.51 g of propylene glycol is added and the resultingmixture is swirled and is sonicated to mix and dissolve the solids. 1.48g of polysorbate 80 is added and is swirled to mix. 0.50373 g of citricacid is added and is swirled to mix. Some citric acid solids remainundissolved. Approximately 10 mL of distilled water is added and isswirled to mix/dissolve the solids. The mixture is diluted to 50 mL withdistilled water and is mixed well such that all solids are in solutionto formulate a liquid having the following pioglitazone concentration ofabout 15 mg/50 mL or 0.3 mg/mL.

In practicing the methods of the present invention, a selected low dosepioglitazone can be administered to a subject using the pioglitazoneliquid of this Example 4. For example, 5 mL or a teaspoonful willdeliver a dose of about 1.5 mg pioglitazone HCl, whereas as 15 mL or atablespoonful will deliver a dose of about 4.5 mg of pioglitazone HCl.Two tablespoonfuls or about 30 mL of the pioglitazone liquid of thisExample 4 will deliver about 9 mg of pioglitazone HCl per dose.

Example 5 Pioglitazone Liquid Formulation 2

A liquid formulation of pioglitazone is prepared using the materials asfollows.

Materials:

Citric Acid, Sigma, C1857, lot 089K0057

Distilled Water, Ice Mountain

HPMC, USP, Sigma, H-3785, lot 122K0149

Pioglitazone HCl, Takeda, lot 345

Polyethylene Glycol 200, Sigma, P3015, lot 098K0056

Polysorbate 80, NF, Spectrum, P0138, lot XV0879

Propylene Glycol, USP/FCC, Fisher, P355, lot 080676

Sucrose, USP, Sigma, S3929, lot 086K0022

Syrup NF, Spectrum, SY105, lot XP0703

Approximately 0.01613 g of pioglitazone HCl is added to a 50-mLvolumetric flask. 1.0043 g of citric is acid is added. Approximately 25mL of distilled water is added and the resulting mixture is swirled andis sonicated to wet the solids. The mixture is diluted to volume, i.e.,about 50 mL, with distilled water, is mixed well and then is sonicatedfor 1-2 minutes such that all solids are in solution.

The liquid pioglitazone solution of this Example 5 will have thefollowing pioglitazone concentration of about 16.13 mg/50 mL or 0.326mg/mL.

In practicing the methods of the present invention using the liquidpioglitazone solution of this Example 5, a selected low dosepioglitazone can be administered to a subject. For example, 5 mL or ateaspoonful will deliver a dose of about 1.63 mg pioglitazone HCl,whereas as 15 mL or a tablespoonful will deliver a dose of about 4.89 mgof pioglitazone HCl. Two tablespoonfuls or about 30 mL of thepioglitazone liquid of this Example 5 will deliver about 9.78 mg ofpioglitazone HCl per dose.

Example 6 Pioglitazone Suspension Formulation 1

A suspension formulation of pioglitazone is prepared as follows.

Preparation of Pioglitazone HCl Suspension A: Suspending Vehicle isSyrup NF (density of Syrup NF is 1.30 g/mL).

0.025 g of Pioglitazone HCl Drug Substance is transferred into a glassmortar and pestle. The Pioglitazone HCl is wetted with about 4 drops ofthe Suspending Vehicle and mixed/ground for about 1 minute to form asmooth uniform paste. The suspending vehicle is added until the totalweight in the mortar and pestle is about 1 g. The resulting mixture ismixed/ground for 1 minute. More suspending vehicle is added until thetotal weight is about 8 g. The resulting mixture is mixed for 1 minute.More suspending vehicle is added until the total weight is about 48 gand then mixed for 1 minute. Suspending Vehicle is added until the totalweight of the suspension is 130.04 g and mixed for 1 minute. The mixturefrom the mortar is poured into a 4 oz reagent bottle. The bottle iscapped and the suspension is shaken by hand for about 1 minute.

The theoretical concentration of pioglitazone HCl is determined;

25.60 mg/130.04 g=0.1969 mg/g (as the HCl salt—not the free base)25.60 mg/100 mL=0.2560 mg/mL (as the HCl salt—not the free base)

In practicing the methods of the present invention using the liquidpioglitazone suspension 1 of this Example 6, a selected low dosepioglitazone can be administered to a subject. For example, 5 mL or ateaspoonful will deliver a dose of about 1.28 mg pioglitazone HCl,whereas as 15 mL or a tablespoonful will deliver a dose of about 3.84 mgof pioglitazone HCl. Two tablespoonfuls or about 30 mL of the liquidpioglitazone suspension 1 of this Example 6 will deliver about 7.68 mgof pioglitazone HCl per dose.

Example 7 Pioglitazone Suspension Formulation 2 Preparation ofSuspending Vehicle B: 0.6% HPMC+10% Sucrose 0.6% HPMC Solution:

1000 mL of distilled water is transferred into a 2-L Erlenmeyer flask.The water is heated to 60° C. with constant stirring. 6 g of HPMC isweighed and is dispersed uniformly into the heated water. Heating of themixture is continued until it just reaches boiling. The mixture isremoved from the heat and is placed in an ice bath with constantstirring. The mixture is stirred until it clarifies and cools to roomtemperature.

Suspending Vehicle: (0.6% HPMC with 10% Sucrose):

80 g of sucrose is added to a 1000-mL glass bottle. 50 mL of distilledwater is added and the mixture is mixed by shaking such all of thesolids are dissolved. 0.6% HPMC Solution is added until the total weightis 800 g. The mixture is shaken to dissolve the solids.

The density of the solution is 103.86 g/100 mL.

Preparation of Pioglitazone HCl Suspension B: Suspending Vehicle is 0.6%HPMC +10% Sucrose

0.025 g of Pioglitazone HCl Drug Substance is transferred into a glassmortar and pestle. The Pioglitazone HCl is wetted with about 4 drops ofthe Suspending Vehicle and is mixed/ground for about 1 minute to form asmooth uniform paste. Suspending vehicle is added until the total weightin the mortar and pestle is about 1 g. The mixture is mixed/ground for 1minute. Additional suspending vehicle is added until the total weight isabout 8 g and then mixed for 1 minute. Additional suspending vehicle isadd until the total weight is about 20 g and then is mixed for 1 minute.

Additional suspending vehicle is added until the total weight is about40 g-50 g and then is mixed for 1 minute. Suspending Vehicle is addeduntil the total weight of the suspension is 103.31 g and is mixed for 1minute. The mixture is poured from the mortar into a 4 oz reagentbottle. The bottle is capped and the suspension is shaken by hand forabout 1 minute.

The theoretical concentration of pioglitazone HCl is determined;

26.44 mg/103.31 g=0.25593 mg/g (as the HCl salt—not the free base)26.44 mg/100 mL=0.2644 mg/mL (as the HCl salt—not the free base)

In practicing the methods of the present invention using the liquidpioglitazone suspension 2 of this Example 7, a selected low dosepioglitazone can be administered to a subject. For example, 5 mL or ateaspoonful will deliver a dose of about 1.322 mg pioglitazone HCl,whereas as 15 mL or a tablespoonful will deliver a dose of about 3.966mg of pioglitazone HCl. Two tablespoonfuls or about 30 mL of the liquidpioglitazone suspension 2 of this Example 7 will deliver about 7.932 mgof pioglitazone HCl per dose.

Example 8

Heretofore there has not been an ability to predict which people aremore likely to develop pathophysiological changes of the kind describedherein that lead to cognitive impairment and ultimately Alzheimer'sdementia. The TOMM40 rs10524523 genotype along with age and possiblyother factors constitute a prognostic biomarker to determine whichsubjects are at risk for developing cognitive impairment of theAlzheimer's type in the next 5-7 years, and thus provide the opportunityfor medical intervention in the early phase of this progressive anddevastating disease. The clinical benefit of this intervention may beconfirmed in a clinical study of the general form described below. Inaddition, a prospective clinical study of this nature would providesufficient data to determine the positive predictive and negativepredictive values of the prognostic biomarker, an understanding of whichis needed prior to introduction of the biomarker into clinical practice.

OPAL Study

rs10524523 (523) is a poly-T length polymorphism that occurs in linkagedisequilibrium (LD) with APOE genotypes, and is inherited together withthe APOE genotype on each strand in the LD region. Essentially a singleintronic variant of TOMM40 varies by poly-T length, with the longerforms of the variant associated with approximately a 7 year differencein the age of onset compared to the shorter forms. Based on thepresenting age of the normal subject, a determination of ‘High risk’ ofonset of cognitive impairment and AD over the next 5-7 years, or ‘Lowrisk’ is determined.

This study provides a novel genetically-based model for theidentification of subjects in large diverse community-based populationswho are at higher risk of AD onset within 5-7 years by combiningclinical risk assessments based on the presence of specific genotypesrelated to Alzheimer's disease onset and clinical expression. The study:

-   -   uses the TOMM40 rs10524523 (523) poly-T length polymorphisms in        the TOMM40-APOE LD region, perhaps in conjunction with the APOE        genotype, for predicting the age of onset of cognitive        impairment and Alzheimer's disease. Specifically, to determine        if a discrete Alzheimer's disease diagnostic test can separate        subjects into ‘High-risk’ and ‘Low-risk’ groups for Alzheimer's        disease; and    -   uses a low dose PPARγ Agonist daily for 60 months (5 years)        versus placebo in pre-symptomatic subjects who are at High risk        as defined by their TOMM40-APOE genotype of Alzheimer's disease,        to delay the onset of Alzheimer's disease related dementia        symptoms.

Cognitively normal subjects between the ages of 62-87 are evaluated forsusceptibility to AD within the next 5-7 years and are tested foreffects of pioglitazone on onset of AD. The definition cognitivelynormal is calculated as within 1.5 standard deviations (SD) of thepopulation mean taking into account the age of the subject and the levelof education for the assessments listed below. Scores below this cut offare considered cognitively impaired. The following cognitive assessmentsare used to assess cognitive function at enrollment and throughout thecourse of the study.

The cognitive assessment scales are chosen to be sensitive to earlydeficits in Alzheimer's disease. These assessment scales are used in theADAPT study (1), which is a prevention study for Alzheimer's diseaseusing NSAID therapy carried out in 2004. The Mini Mental examination(2MS-E) is used in the Women's Health Initiative Study for hormonereplacement therapy (2) for the prevention of AD. Thus, the cognitiveassessments include:

-   -   Modified Mini Mental State Examination (3MS-E)    -   Brief Visuospatial Memory Test (Revised) (BVMT-R)    -   Hopkins Verbal Learning Test (Revised) (HVLT-R)    -   Rivermead Behavioural Memory Test (RBMT)    -   Generative Verbal Fluency Test (GVFT)    -   Digit Span Test (DST)

Enrollment into the study is based solely on the scores from theseassessments. For randomization into the study, the individuals inaddition are given a DNA test consisting of APOE genotyping andmeasurement of the 523 poly-T repeat lengths to assess their risk statusas ‘High risk’ or ‘Low risk’ of developing cognitive impairment or ADover the next 5-7 years. The following designs describe the studyprocedure

Study Design Assumptions

The end points are 1) change in a measure of cognition from baselinebased on the scores from the neuropsychological assessments and 2)diagnosis of Alzheimer's disease in accordance with NINCDS-ADRDAcriteria (National Institute of Neurological and Communicative Disordersand Stroke (NINCDS) and Alzheimer's Disease and Related DisordersAssociation (ADRDA). These are either taken as two primary end points ora combined event end point.

Sample Size Calculations

The sample size calculation is determined for a log-rank test of time toevent data based on the above end points. It is assumed that theconversion rate for the ‘High risk’ group will be 20% at the end of 5years follow-up based on data from previous prevention studies (3,4). Asample size of 374/group is required to detect a 50% improvement in thisconversion rate (i.e. from 20% to 10%) at the 5% level of significanceand with 90% power. A drop out rate of 20% for both placebo andtreatment groups over the five year period is built into thiscalculation. This sample size is not adjusted for multiple comparisons.

A further assumption is made that the ‘Low risk’ group has a conversionrate of 10% based on incidence rates of Alzheimer's disease in thegeneral population (4). The sample size required to compare this groupwith the ‘High risk’ placebo group is again 374/group with 90% power anda 5% significance level.

Study Designs

The diagnostic test defines which patients are at ‘High risk’ ofconversion to Alzheimer's disease or cognitive impairment, (High risk)and which patients are at ‘Low risk’ of conversion (Low risk). Theinvestigators are blinded to the results of the diagnostic test andcentral randomization is used to maintain this blind. The mainobjectives for any design are:

-   -   to determine whether the diagnostic test can discriminate        between ‘High’ and ‘Low risk’ subjects, and    -   to evaluate the effect of treatment on the conversion rate of        ‘High risk’ patients.

All subjects recruited for these studies will be cognitively normal asdefined previously.

Preferred Study Design

In this design, only the ‘High risk’ group is randomized to receiveplacebo or treatment. This is a simple design that, for example,utilizes a total sample size of 1122 subjects. This design allows twohypotheses to be investigated: the first relates to the ability of thediagnostic to define the ‘High’ and ‘Low risk’ groups by comparing thedata from the placebo treated subjects; the second relates to whetherthe treatment can improve the conversation rate by comparing the datafrom the treatment and placebo groups of the ‘High risk’ arm.

Alternative Design 1

In this design a fourth group is added to allow the effect of treatmentto be evaluated in the ‘Low risk’ group. This design may increase thetotal sample size to 1496 patients.

This design may provide useful information if the ‘Low risk’ group has ahigher than expected conversion rate. However, there are potentialconcerns with this design in terms of risk/benefit to the ‘Low risk’group. Subjects in the ‘Low risk’ group might be at risk of experiencingside effects with treatment with no expected benefit to their conversionrate.

Alternative Design 2

This design is the same as the preferred design except that the ‘Lowrisk’ group remains untreated and serves as an observational group. Thisdesign will be able to meet the objectives of the study but there are anumber of potential pitfalls:

-   -   unable to blind the untreated arm so results of the diagnostic        test will not be blinded,    -   possibly higher drop out rate in the ‘Low risk’ group if        subjects feel that being observed but not ‘treated’ is without        benefit.    -   will not be comparing like with like which could be an issue if        there is a ‘placebo’ effect (unlikely for time to event but        possible for cognitive testing).

The sample size calculations are based on detecting a difference of 10percentage points between conversion rates at the end of 5 years. Anincrease in numbers allows a signal to be detected earlier with asmaller difference. If it is assumed that the conversion rate in the‘High risk’ group is 5%/year then after three years approximately 15% ofthe subjects may have converted to Alzheimer's disease or show cognitiveimpairment. Assuming that treatment can improve this rate by 50% thenthe expected conversion rate in the treated group will be 7.5%. In orderto detect the difference with 90% power an the 5% significance, 559subjects per group will be required resulting in a total sample size forthe preferred design of 1677. This increase in subject numbers permitsinvestigation of a family of age of onset curves associated with eachTOMM40-APOE haplotype. An exploratory analysis is used to investigatethe effects of age by including age as a covariate in a Cox'sproportional hazards time to event analysis, which allows theinvestigation of covariates. A certain percentage of subjects aredefined as having mild cognitive impairment (MCI) based on theneuropsychological assessments at screening. The study will only recruitthose subjects who are defined as cognitively normal based on theneuropsychological assessments.

REFERENCES

-   1) ADAPT Research Group: Cognitive Function Over Time in the    Alzheimer's Disease Anti-inflammatory Prevention Trial (ADAPT)    Results of a Randomized, Controlled Trial of Naproxen and Celecoxib:    Archives of Neurology, Vol 65 (No 7), July 2008-   2) Stephen R. Rapp; Mark A. Espeland; Sally A. Shumaker et al:    Effect of Estrogen Plus Progestin on Global Cognitive Function in    Postmenopausal Women, The Women's Health Initiative Memory Study:    2003; 289(20):2663-2672 JAMA-   3) Curtis L. Meinert, John C. S. Breitner: Chronic disease long-term    drug prevention trials: Lessons from the Alzheimer's Disease    Anti-inflammatory Prevention Trial (ADAPT): Alzheimer's & Dementia    4 (2008) S7-S14-   4) Stephen Salloway, Stephan Correia: Alzheimer disease: Time to    improve its diagnosis and treatment: Cleveland Clinic Journal Of    Medicine Volume 76, Number 1 Jan. 2009-   5) ADAPT Research Group: Cognitive Function Over Time in the    Alzheimer's Disease Anti-inflammatory Prevention Trial (ADAPT)    Results of a Randomized, Controlled Trial of Naproxen and Celecoxib:    Archives of Neurology, Vol 65 (No 7), July 2008-   6) Stephen R. Rapp; Mark A. Espeland; Sally A. Shumaker et al:    Effect of Estrogen Plus Progestin on Global Cognitive Function in    Postmenopausal Women, The Women's Health Initiative Memory Study:    2003; 289(20):2663-2672 JAMA-   7) Curtis L. Meinert, John C. S. Breitner: Chronic disease long-term    drug prevention trials: Lessons from the Alzheimer's Disease    Anti-inflammatory Prevention Trial (ADAPT): Alzheimer's & Dementia    4 (2008) S7-S14-   8) Stephen Salloway, Stephan Correia: Alzheimer disease: Time to    improve its diagnosis and treatment: Cleveland Clinic Journal Of    Medicine Volume 76, Number 1 Jan. 2009

Example 9 BOLD Study

The invention provides for the following exemplary dose findinganalysis.

The invention provides for measuring pharmacodynamic changes in responseto different low doses of pioglitazone. The pharmacodynamic measure thatis relevant is a change in regional blood oxygenation coupled toneuronal activity as measured by blood oxygen level dependent functionalmagnetic resonance imaging (BOLD fMRI).

Neuroprotection and mitochondrial biogenesis are among the physiologicaleffects of thiazolidinediones. In one embodiment, pioglitazone treatmentof subjects may increase the metabolic capacity of active regions of thebrain. This change in metabolic capacity may be observable using BOLDfMRI.

BOLD fMRI is a widely used technology for non-invasive whole brainimaging. This technique measures a change in regional blood oxygenationcoupled to neuronal activity.

BOLD fMRI measures the relative change in the ratio of oxy-todeoxyhemoglobin in the brain that occurs as a result of neuronalactivity. As neurons become active, there is a concomitant increase incell metabolism, and blood flow increases to regions of increasedneuronal activity to meet these metabolic demands. The result of thishemodynamic response is a measurable change in the local ratio of oxy-todeoxyhemoglobin. Oxyhemoglobin is diamagnetic and deoxyhemoglobin isparamagnetic and this difference in magnetism is detected by BOLD fMRI.

BOLD signals reflect complex and incompletely understood changes incerebral blood flow (CBF), cerebral blood volume (CBV) and cerebralmetabolic rate of oxygen consumption (CMRO₂) following neuronalactivity. Candidate circuit elements for triggering various kinds ofBOLD signals include excitatory neurons, mixed neuronal populations,astroglia, and axonal tracts or fibres of passage (described in detailin Lee et al., 2010 Nature 465: 788-792; Logothetis 2008 Nature 453:869-878; Logothetis et al. 2001 Nature 412: 150-1571; Raichle 2010 Cell14: 180-190, each of which is incorporated herein by reference in itsentirety).

The study will utilize healthy, cognitively normal, older subjects ofthe age of interest, e.g., between 62 and 87. BOLD fMRI scanning will beperformed using a scanner optimized for high-resolution structural andfunctional brain imaging (for example a state-of-the-art GE 3 Teslascanner).

In one embodiment, the study is a double-blinded study using multiplecohorts, with each cohort receiving a different pioglitazone dose. Inanother embodiment, the study is of a serial design wherein the samecohort receives multiple different drug doses. The pharmacodynamicmarker used to indicate changes in neuronal activity as a result ofexposure to pioglitazone is a change in BOLD signal, especially in thedorsolateral prefrontal cortex and hippocampus which are associated withthe higher cognitive functions that are impaired in Alzheimer's disease.

Each participant will undergo MRI scanning on at least three occasions:

1. pre-dose (to obtain a baseline or control value for each subject);2. soon after receipt of the first dose (either at 2 hours or theapproximate time of Cmax) to measure the result of acute exposure todrug; and3. following 7 days of drug exposure (when pioglitazone serumconcentration should each steady state and physiological effect of drugon mitochondrial function should have occurred).

Pioglitazone will be given every day for 7 days.

45 mg of pioglitazone (the marketed formulation for the treatment oftype 2 diabetes) results in a Cmax of approximately 3 mM in serum (seeGhosh et al. 2007 Mol. Pharmacol 71: 1695-1702).

The test doses include:

a) 0.5 mg dose-approximately 33.3 nM serum and approximately 6.7 nMbrain

b) 1.5 mg dose-approximately 100 nM serum and approximately 20 nM brain;

c) 4.5 mg dose-approximately 300 nM serum and approximately 50 nM brain;

d) 9 mg dose-approximately 600 nM serum and approximately 120 nM brain.

Magnetic Resonance Imaging Protocol Summary

General Participant Screening Procedure

Participants will be screened for ferrous metal implants that wouldpreclude scanning prior to selection. Participants will be instructed tofast and abstain from caffeine, tobacco products and exercise for twohours prior to the scan session, and refrain from drinking alcohol andtaking non-essential medication for twelve hours prior to scanning.Participants taking stimulant medications will be asked not to take themfor at least 24 hours with physician approval. Two breath samples willbe obtained to measure alcohol levels. Urine samples will be obtained totest for 5 drug metabolites (psychostimulants, cannabis, opiates andsedatives). Female participants will be given a urine pregnancy test,which must be negative for the participant to undergo scanning.

General Scanning Protocol

Subjects will be provided the opportunity to enter an MRI simulator toassess their comfort level for participating in the MRI session.Participants will then be instrumented for heart rate(photoplethysmograph) and blood pressure monitoring and will bepositioned in the scanner. Head movement will be minimized using acombination of pillows and tape. After acquiring localizer scans, theprotocols will be presented in the following fixed order, with a totalscan time of approximately 60 minutes.

Structural MRI.

Measures of total and regional gray and white matter as well as CSF willbe collected using high resolution MRI.

Technical Details: T1-weighted images with 1 mm isometric voxels will beacquired using the Array Spatial Sensitivity Encoding Techniques (ASSET)with fast spoiled gradient-recall (FSPGR). Image parameters will beoptimized for contrast between white matter, gray matter and CSF(TR/TE/flip angle=7.484 ms/2.984 ms/12°, 256 mm FOV, 1 mm slice, 166slices, 256×256 matrix, 1 Nex).

Perfusion MRI.

Measures of total and regional resting cerebral blood flow will becollected using Pulsed Arterial Spin Labeling (PASL).

Technical Details: Interleaved images with and without labeling will beacquired using a gradient echo-planar imaging (EPI) sequence.Acquisition parameters consist of the following: field of view (FOV)=22cm, matrix=64×64, repetition time (TR)=3 sec, echo time (TE)=17 msec,label time=1.6 sec, delay time=0.8 sec, flip angle=90°. The restingperfusion scanning protocol takes approximately 6 minutes during whichsubjects will be instructed to lie still ad let their minds go blank,but keep their eyes open and stay awake. Data corresponding to fourteenslices (8 mm thickness with 2 mm gap) will be acquired in sequentialorder from inferior to superior.

Functional MRI (fMRI).

Archival working and episodic memory stimulation paradigms will beadministered to measure patterns of neural activation, especially in thedorsolateral prefrontal cortex and hippocampus, associated with highercognitive functions impaired in Alzheimer's disease using blood oxygenlevel-dependent (BOLD) fMRI.

Technical Details: A series of 34 interleaved axial functional sliceswill be acquired for full-brain coverage (TR/TE/flip=2000/31/60; FOV=240mm; 3.75×3.75×3.8 mm voxels; interslice skip=0) using an inverse-spiralpulse sequence to reduce susceptibility artifact. High-resolutionthree-dimensional spin-echo co-planar structural images will be acquiredin 68 axial slices (TR/TE/flip=12.2/5.3/20, voxel size=1×1×1.9 mm,FOV=240 mm, interslice skip=0) for normalization and subject averaging.

fMRI Stimulation Paradigms Working Memory; See Mattay et al., PNAS 2003for details. Episodic Memory: See Bookheimer et al. New England Journalof Medicine 2000 for details.

Example 10 Rat BOLD Study

Low dose pioglitazone penetrates the blood brain barrier and induceschanges in brain physiology.

It was determined whether low doses of pioglitazone HCl penetrate theblood brain barrier in sufficient concentrations to elicit functional ormolecular changes in the brain. BOLD fMRI was used to measuredrug-related changes in resting state functional connectivity across thewhole brain.

Adult male Wistar rats (275±25 g) were housed separately and maintainedon a 12-h light,12-h dark schedule. Food and water was provided adlibitum. Animals were cared for in accordance with the guidelinespublished in the Guide for the Care and Use of Laboratory Animals(National Institutes of Health Publications No. 85-23, Revised 1985).Animal body weights were measured approximately 24 hours before Day-3,and on Study Days 3 and 6.

Pioglitazone HCl (PIO) was dissolved in 0.5 mol/L citric acid (CA) toyield a stock solution at a concentration of 0.32 mg/10 mL/kg. Otherdosages were prepared by appropriate dilution of the stock solution with0.5 mol/L CA to yield dose volumes of 10 mL/kg. Control rats receivedthe vehicle at 10 mL/kg. Dose concentrations were based on the weight ofthe test article as supplied (i.e., as the HCl salt), with the doseadjusted to the most recent body weight of the animal. Daily dosing withPIO in solution was by oral gavage at approximately the same time everyday. Animals were anesthetized lightly with isoflurane immediatelybeforehand to facilitate dosing.

All animals used in the imaging studies were acclimated to the MRIholding device by being placed in it for 15-90 minutes daily for atleast 7 days, as previously described (Zhang et al. 2010 J NeurosciMethods 189: 186-196; Liang et al. 2011 J Neurosci 31: 3776-3783).

After the acclimation period, animals were assigned to 1 of 7 treatmentarms matched for mean body weights (see Table 1). Dosing occurred oncedaily, at approximately the same time every day. All animals were imagedat Baseline (Study Day-3), approximately 2.5 to 3 h after dosing withvehicle. Dosing began 3 days later (Study Day 1). On this day, allanimals were administered either vehicle (CA) or PIO depending on theirgroup assignment. On Study Day 2, one vehicle group and one grouptreated with PIO at 0.08 mg/kg/day (Acute Arm) were imaged approximately2.5 to 3 h after dosing. For all groups, dosing continued for seven daystotal. On Study Day 7, all rats were imaged approximately 2.5 to 3 hafter administration of the final dose.

TABLE 1 Treatment Arms, Daily PIO Dose and Imaging Time Points DailyDose Imaging Time-Points Treatment (mg/kg) Study Day −3 Study Study Arm(N = 5/group) (Baseline) Day 2 Day 7 Acute 0, 0.08 ✓ ✓ ✓ Sub-chronic 0,0.04, 0.08, ✓ No imaging ✓ 0.16, 0.32

Extrapolation to the corresponding dosage in humans was achieved whileadjusting for the relative AUC for each. In humans, a dose of 7.5 mg isassociated with an AUC of 2.8 μg·h/mL. In rats, a dose of 0.50 mg/kg/dayPIO HCl is associated with an AUC of 7.11 μg·h/mL. Results of thesecalculations are presented in Table 2.

TABLE 2 Rat and Human-Equivalent Doses, Based on Extrapolated ExposuresHuman Dose (mg total/day) Parameter 1.5 3 6 12 Rat Dose 0.04 0.08 0.160.32 (mg/kg/day) Targeted 0.57 1.14 2.28 4.55 AUC (μg · h/mL)

Animal preparation activities related to imaging were initiated toinsure that the imaging itself occurred approximately 2.5 to 3 h afterdosing. The animals were prepared for positioning in the restraint underisoflurane anesthesia as previously described (Zhang et al. 2010,supra). This procedure took approximately 10-15 minutes, by which timeanimals were usually fully conscious. Imaging was conducted on awakeanimals.

All MR experiments were conducted using a 4.7 T/40 cm horizontal magnet(Oxford, UK) interfaced with a BiospecBruker console (Bruker, Germany)and equipped with a 20 G/cm magnetic field gradient. A dual ¹Hradiofrequency (RF) coil configuration (Insight Neurolmaging Systems,Worcester, Mass.) consisting of a volume coil for exciting the waterproton spins and a surface coil for receiving MRI signal was used; thevolume and surface coils were actively tuned and detuned to preventmutual coil coupling. This dual-coil configuration allowed forsufficient RF field homogeneity in the rat brain for RF transmission,while preserving the advantage of higher signal-to-noise ratio (SNR)provided by the smaller reception coil.

Anatomical images were acquired first using a multi-slice fast spin-echosequence (RARE) with the parameters: repetition time (TR)=2125 ms; RAREfactor=8; effective echo time (TE)=50 ms; matrix size=256×256; field ofview (FOV)=3.2×3.2 cm²; slice number=18; slice thickness=1 mm; n=8.Based on the geometry of anatomical images, multi-slice gradient-echoimages covering the whole brain were acquired using echo-planar imaging(EPI) with the parameters: TR=1 s; Flip Angle=60°; TE=30 ms; matrixsize=64×64; FOV=3.2×3.2 cm²; slice number=18; slice thickness=1 mm. Ratswere at rest during image acquisition. 200 volumes were acquired foreach run; 9 runs were obtained for each rat.

Analysis of all fMRI data was conducted using Medical ImageVisualization and Analysis (MIVA), Statistical Parametric Mapping (SPM8)software (Wellcome Department of Cognitive Neurology, London, UK) andMatlab (The Mathworks Inc., Natick, Mass., USA). The data was initiallycorrected for motion (threshold of 0.25 mm). Further pre-processing ofthe data included (a) slice scan time correction, (b) spatial smoothingusing a 3D Gaussian filter (1-mm FWHM) to account for small variationsin signal due to movement and vascular effects, and (c) voxel-wiselinear detrending and high-pass filtering of frequencies (3 cycles pertime course) to adjust for scanner drift between runs. Structural andfunctional data of each animal was then transformed to standardstereotaxic space embedded in MIVA to facilitate group analysis offunctional data.

Correlational functional connectivity analysis was used to analyzeresting-state functional connectivity. First, each animal was alignedand co-registered, based on anatomical images, to a fully segmented ratbrain atlas. The co-registration procedure will provide the coordinatesof each seed region of interest (ROI) in the image space. Afterco-registration and alignment, fMRI time courses for individual voxelsin a seed ROI were obtained according to their correspondingcoordinates. A time course for each seed region was created byregionally averaging time courses from all pixels inside the seed ROI.All ROI time courses were 0.002-0.08 Hz band-pass filtered. Afterfiltering, the Pearson cross-correlation (CC) coefficient between ROItime courses was calculated and used to quantify the strength offunctional connectivity.

To evaluate the effects of PIO on functional connectivity across thewhole brain, we divided the whole rat brain into 57 ROIs. The strengthof functional connectivity between each pair of ROIs was evaluated usingthe cross-correlation coefficient between the two ROI time courses. Intotal 57×5612=1596 functional connections were assessed for each rsfMRIruns. This procedure was repeated for all 9 runs of each fMRI sessionand the connectivity strength of the corresponding connection was thenaveraged across 9 runs. As a result, the connectivity strength of 1596connections was obtained for each rsfMRI scan session.

For each connection (i.e. a connection between each pair of ROIs),repeated measure ANOVA with the factors of imaging day, dosage andinteraction were then calculated. Statistical significance level was setat P<0.005, uncorrected.

To evaluate the effects of PIO on the individual neural circuitries,seed-based correlational analysis was used (Zhang et al. 2010, supra).The CA1 of the hippocampus was selected as the seed ROI. The spatialpattern of brain regions that are functionally connected with the seedROI was calculated in a voxel-by-voxel manner. First, the regionallyaveraged time course of the seed ROI was obtained as a reference.Cross-correlation coefficient between the time course of each voxel andthe reference time course was then calculated. The correlationcoefficient represented the functional connectivity strength betweenthis voxel and the seed. A connectivity map for the seed ROI was createdfor each fMRI run and maps across nine runs were then averaged to createthe connectivity map for each scan session. At last, a compositeconnectivity map was generated by averaging connectivity maps acrossrats of the same group that were imaged on the same day in the protocol(Zhang et al. 2010, supra).

FIG. 1 provides an example of the fMRI data and demonstrates that eventhe lowest doses of orally-administered, immediate release pioglitazoneproduce a change in metabolism in the central region of the deepcortical structures of the brain. This is consistent with anintracellular mitochondrial effect

CONCLUSIONS

-   -   1. Relative to vehicle control, there is evidence that PIO        treatment at doses as low as 0.04 mg/kg/day induces changes in        multiple brain regions in the rat. This result indicates that        low-dose PIO, administered orally, penetrates the blood brain        barrier.    -   2. PIO, at doses as low as 0.08 mg/kg/day, induced functional        changes as early as 24 hours, which was the earliest time point        assayed after initiation of treatment.

As seen in FIG. 1, there appears to be a diminished signal at the 0.32mg/kg/day dosage based on the appearance of these data. However, furthertesting will be done in order to confirm whether or not there is anactual diminished effect at this dosage relative to the lower dosages,and not simply reflecting intrinsic biological variability between theanimal subjects.

Example 11 Exemplary Risk Determination

In order to identify subjects having normal cognition who are at highrisk of developing cognitive impairment of the Alzheimer type (alsotermed hippocampal type) in the next 5 years based on TOMM40 rs1054523(523) genotype, age, and possibly APOE genotype, age-of-onset data werestudied from a cohort of 438 prospectively followed individuals from theDuke Bryan ADRC Memory Health and Aging study.

Table 3 summarizes an exemplary risk categorization based on 523 andAPOE genotypes and age. Note that there appears to be a subset of VL/VL,APOE ε3/ε3 subjects who succumb to the onset of Alzheimer's diseasebetween the ages of 51 and 59. These subjects are not considered inTable 3, which presents only the low risk subset of VL/VL carriers whoare cognitively normal after age 62. An expanded risk categorizationthat includes the younger ‘high risk’ VL/VL APOE ε3/ε3 subjects is alsocontemplated.

TABLE 3 Exemplary Age Thresholds That Define High Risk for 523 Genotypesat Ages 62-83 523 or APOE Genotype Age defining high risk 523 L, L Allhigh risk 523 L, VL All high risk 523 S, L 74 523 S, S 77 523 S, VL 76523 VL, VL All low risk APOE ε2/ε2 All low risk APOE ε2/ε3 All low riskAPOE ε2/ε4 All low risk

An exemplary use of these assignments is straightforward. Table 3 isused to make assignments of individuals into the high- or low-riskgroups (which may be irrespective of ethnicity) as follows:

-   -   1) individuals with a 523 genotype of (L,L) or (L,VL) are        assigned to the high-risk group,    -   2) individuals with a 523 genotype of (VL,VL) (>62 years) or        APOE genotype of (c2/c2) or (c2/c3) are assigned to the low-risk        group,    -   3) for individuals with a 523 genotype of (S,S), (S,VL) or        (S,L), an individual's current age is compared to the age in        Table 2 to make the risk assignment.

For each 523 genotype, the corresponding age-of-onset curve forcognitive impairment is examined to identify the age where the slope ofthe curve indicates high risk of development of cognitive impairment ina 5-year window. The steep portion of the curve follows a relativelyflat asymptote and has a characteristic time point (age) where a rapidincrease in the proportion of individuals with cognitive impairment isobserved (see FIG. 2 and FIG. 3).

FIG. 3 illustrates determination of an age used to distinguish high- andlow-risk classification for the (S,L) 523 genotype. The steep part ofthe curve can be identified as starting at about age 74, whichcorresponds to the age associated with a level of 90% of individualswith this genotype not presenting with cognitive impairment. Therefore,individuals aged 74 or older may be assigned to the high-risk group forthe study, whereas individuals younger than 74 are assigned to thelow-risk group. Exemplary age-of-onset curves for cognitive impairmentfor the remaining 523 genotypes are provided in FIGS. 4-9, which arereflected in the assignments in Table 2 presented above.

It should be understood that, while the graphs presented herein areinterpreted to give a specific age where the slope change occurs, thesegraphs may be updated as additional data are collected to modify and/oroptimize the age designations without departing from the generalteachings of this method.

The disclosures of the patents, patent documents, articles, abstractsand other publications cited herein are incorporated by reference hereinin their entireties as if each were individually incorporated. In caseof conflict, the present specification, including definitions, shallcontrol. Various modifications and alterations to this invention willbecome apparent to those skilled in the art without departing from thescope and spirit of this invention. Illustrative embodiments andexamples are provided as examples only and are not intended to limit thescope of the present invention. The scope of the invention is limitedonly by the claims set forth as follows.

1.-40. (canceled)
 41. A method of delaying the onset of Alzheimer'sdisease in a subject at risk of developing Alzheimer's diseasecomprising: a. determining the presence of at least one genetic variantof the TOMM40 gene, wherein said genetic variant is an rs10524523allele, and wherein the presence of said at least one genetic variantindicates a risk of developing Alzheimer's disease; and b. administeringan effective amount of low dose pioglitazone or a salt thereof to saidsubject thereby delaying the onset of Alzheimer's disease in saidsubject.
 42. A method of delaying the onset of mild cognitive impairmentin a subject at risk of developing Alzheimer's disease comprising: a.determining the presence of at least one genetic variant of the TOMM40gene, wherein said genetic variant is an rs10524523 allele, and whereinthe presence of said at least one genetic variant indicates a risk ofdeveloping Alzheimer's disease; and b. administering an effective amountof low dose pioglitazone or a salt thereof to said subject therebydelaying the onset of mild cognitive impairment in said subject.
 43. Amethod of delaying the onset of amnestic mild cognitive impairment in asubject at risk of developing Alzheimer's disease comprising: a.determining the presence of at least one genetic variant of the TOMM40gene, wherein said genetic variant is an rs10524523 allele, and whereinthe presence of said at least one genetic variant indicates a risk ofdeveloping Alzheimer's disease; and b. administering an effective amountof low dose pioglitazone or a salt thereof to said subject therebydelaying the onset of amnestic mild cognitive impairment in saidsubject.
 44. A method of delaying the onset of preclinical Alzheimer'sdisease in a subject at risk of developing Alzheimer's diseasecomprising: a. determining the presence of at least one genetic variantof the TOMM40 gene, wherein said genetic variant is an rs10524523allele, and wherein the presence of said at least one genetic variantindicates a risk of developing Alzheimer's disease; and b. administeringan effective amount of low dose pioglitazone or a salt thereof to saidsubject thereby delaying the onset of preclinical Alzheimer's disease insaid subject.
 45. A method of delaying the onset of prodromalAlzheimer's disease in a subject at risk of developing Alzheimer'sdisease comprising: a. determining the presence of at least one geneticvariant of the TOMM40 gene, wherein said genetic variant is anrs10524523 allele, and wherein the presence of said at least one geneticvariant indicates a risk of developing Alzheimer's disease; and b.administering an effective amount of low dose pioglitazone or a saltthereof to said subject thereby delaying the onset of prodromalAlzheimer's disease in said subject.
 46. A method of delaying the onsetof physiological changes associated with Alzheimer's disease in asubject at risk of developing Alzheimer's disease comprising: a.determining the presence of at least one genetic variant of the TOMM40gene, wherein said genetic variant is an rs10524523 allele, and whereinthe presence of said at least one genetic variant indicates a risk ofdeveloping Alzheimer's disease; and b. administering an effective amountof low dose pioglitazone or a salt thereof to said subject therebydelaying the onset of physiological changes associated with Alzheimer'sdisease in said subject.
 47. The method of claim 41, whereinpioglitazone is administered at a dosage of 0.5 mg to 9 mg per day. 48.The method of claim 41, wherein pioglitazone is administered at a dosagethat provides an AUC of from about 0.15 μg·h/mL to about 3.6 μg·h/mL.49. The method of claim 41, wherein said administration step comprisesadministering the pioglitazone or a salt thereof to said subject inaccordance with a daily treatment regimen.
 50. The method according toclaim 49, wherein said administration step comprises administering thepioglitazone or a salt thereof to said subject as a pharmaceuticalformulation. 51.-65. (canceled)
 66. The method of claim 41, wherein saidsubject has normal cognition.
 67. The method of claim 41, wherein saidadministration occurs when said subject is younger than
 60. 68. Themethod of claim 41, wherein said administration occurs when said subjectis between the ages of 60 and
 70. 69. The method of claim 41, whereinsaid subject has one copy of the long TOMM40 rs10524523 allele.
 70. Themethod of claim 41, wherein said subject has two copies of the longTOMM40 rs10524523 allele.
 71. The method of claim 41, wherein saidsubject has an increased risk of developing Alzheimer's disease ascompared to a control subject.
 72. The method of claim 71, wherein saidcontrol subject carries at least one copy of the TOMM40 rs10524523allele comprising a poly-T repeat that is less than 19 nucleotides inlength.
 73. The method of claim 71, wherein said control subject doesnot have a copy of the TOMM40 rs10524523 allele comprising a poly-Trepeat that is greater than 19 or greater nucleotides in length.
 74. Themethod of claim 50, wherein the pharmaceutical formulation is abioequivalent formulation.
 75. The method of claim 50, wherein thepharmaceutical formulation is a pharmaceutically equivalent formulation.76. The method of claim 50, wherein the pharmaceutical formulation is atherapeutically equivalent formulation. 77.-81. (canceled)